Novel siglecs and uses thereof

ABSTRACT

The present invention provides proteins, peptide fragments, nucleic acid molecules and fragments thereof, complementary sequences, allelic forms, homologues, antibodies and variants of SIGLEC-BMS sequences, which are new members of the Sialoadhesin subgroup having protein homology to CD33, and methods of using these molecules.

[0001] This application is based on a provisional application, U.S.Serial No. 60/220,139, filed Jul. 21, 2000, the contents of which arehereby incorporated by reference in their entirety into thisapplication.

[0002] Throughout this application, various publications are referenced.The disclosures of these publications are hereby incorporated byreference herein in their entireties.

FIELD OF THE INVENTION

[0003] The present invention relates to sialoadhesin nucleotidesequences, herein designated “SIGLEC-BMS”, and novel SIGLECpolypeptides, and uses thereof.

BACKGROUND OF THE INVENTION

[0004] A group of sialic acid-dependent adhesion molecules has beendescribed within the superfamily of immunoglobulin-like molecules (Kelm,S. et al., 1998 Eur. J. Biochem 255:663-672). The term “Siglec” has beenadopted to describe this family (Sialic acid-binding Ig-relatedlectins). To date, the members of the group include Siglec-1(sialoadhesin), Siglec-2 (CD22), Siglec-3 (CD33), Siglec-4(myelin-associated glycoprotein or MAG), Siglec-4b (Schwann cell myelinprotein or SMP), Siglec-5 (OB-BP2), Siglec-6 (OB-BP1, CD33L), Siglec-7,and Siglec-8 (Table 1).

[0005] The biological activity of the protein members of the Siglecgroup is, for the most part, not understood. However, Siglec proteinsare thought to be involved in diverse biological processes such ashemopoiesis, neuronal development and immunity (Vinson, M. et al., 1996supra). Studies also suggest that these proteins mediate celladhesion/cell signaling through recognition of sialyated cell surfaceglycans (Kelm, S. et al., 1996 Glycoconj. J. 13:913-926; Kelm, S. etal., 1998 Eur. J. Biochem. 255:663-672; Vinson, M. et al., 1996 J. Biol.Chem. 271:9267-9272). TABLE 1 Members of the Siglec protein family andtheir distribution Protein: Expressed on: Recognizes: Target CellReferences: Siglec-1 Macrophage Siaα2,3 Galβ1,3 GalNAc Myeloid cellsCrocker et al. 1194 EMBO J. 13: 4490-4503 (Sialoadhesin) SubpopulationsSiaα2,3 Galβ1,3/4 GlcNAc Neutrophils Crocker et al. 1997 GlycoconjugateJ. 14: 601-609 Vinson et al. 1996 J. Biol. Chem. 271: 9267-9272 Kelm etal. 1998 Eur. J. Biochem. 255: 663-672 Barnes et al. 1999 Blood 93:1245-1252 Siglec-2 B-cells Siaα2,6 Galβ1,4 GlcNAc Lymphocytes Kelm etal. 1998 Eur. J. Biochem. 255: 663-672 (CD22) Siglec-3 Myelomonocyticcells Siaα2,3 Galβ1,3 GalNAc Myelomonocytic Freeman et al. 1995 Blood85: 2005-2012 (CD33) Siaα2,3 Galβ1,3/4 GlcNAc cells Kelm et al. 1998Eur. J. Biochem. 255: 663-672 Taylor et al. 1999 J. Biol. Chem. 27411505-11512 Siglec-4a Oligodendricytes Siaα2,3 Galβ1,3 GalNAc NeuronsKelm et al. Eur. J. Biochem. 255: 663-672 Schwann cells OligodendricytesSiglec-4b Schwann cells (quail) Siaα2,3 Galβ1,3 GalNAc Neurons Kelm etal. 1998 Eur. J. Biochem. 255: 663-672 Siglec-5 Neutrophils Siaα2,3Galβ1,3 GalNAc Erythrocytes Cornish et al. 1998 Blood 92: 2123-2132(OB-BP2) Monocytes Siaα2,6 Galβ1,3 GalNAc Siglec-6 Placenta Not knownNot known Takei et al. 1997 Cytogen. Cell. Genet. 78: 295-300 (OB-BP1,CD33L) B-cells Patel et al. 1999 J. Biol. Chem. 274: 22729-22738Siglec-7 Granulocytes, monocytes, Siaα2,3 Galβ1,3 GalNAc Not knownNicoll et al. 1999 J. Biol. Chem. 274: 34089-34095 (AF170485) NK cells,CD8 + T cells Siaα2,6 Galβ1,3 GalNAc Eosinophils (L3a-604, L3b-595,L3c-995, L3d963) Siglec-8 Eosinophils NAcα2,3 Galβ1,4 Glc Not knownFloyd et al. 2000 J. Biol. Chem. 275: 861-866 (AF 195092)

[0006] The known Siglec proteins are expressed in diverse hemopoieticcell types, yet they all share a similar structure including a singleN-terminal V-set domain (membrane-distal) followed by variable numbersof extracellular C2-set domains, a transmembrane domain, and a shortcytoplasmic tail (FIG. 1). Additionally, the terminal V-set domain hasan unusual intrasheet disulfide bridge that is unique among members ofthe Ig superfamily (Williams, A. F. and Barclay, A. N. 1988 Annu. Rev.Immunol. 6:381-405; Williams, A. F., et al., 1989 Cold Spring HarborSymp. Quant. Biol 54:637-647; Pedraza, L., et al., 1990 J. Cell. Biol.111:2651-2661).

[0007] Results of various research approaches, including truncatingmutants (Nath, D., et al., J. Biol. Chem. 270:26184-26191),site-directed mutagenesis (Vinson, M., et al., 1996 J. Biol. Chem.271:9267-9272; Van der Merwe, P. a., et al., 1996 J. Biol Chem.271:9273-9280), X-ray crystallography and NMR (discussed in: Crocker, P.R., et al., 1997 Glycoconjugate J. 14:601-609) have unequivocallydemonstrated that the GFCC′C″ face of the N-terminal V-set domain ofknown Siglec proteins interact with sialic acid. Thus, the V-set domainmediates cell-to-cell adhesion by interacting with sialic acid. Inparticular, an arginine residue within the V-set domain is a key aminoacid residue for binding to sialic acid (Vinson, M., et al., 1996supra).

[0008] The purported ligands for the known Siglec proteins areglycoproteins or glycolipids on other cells, or in some instances on thesame cell, modified to include sugars or sialic acid (Table 1). Thereare approximately 40 naturally occurring sialic acids (Sia) adding tothe structural diversity of cell surface glycoproteins. The most commonare NeuSAc, Neu9Ac2 and Neu5Gc, occurring in terminal positions linkedto other sugars like Gal, GalNAc, GlcNAc and Sia itself on glycoproteinsand glycolipids. It is postulated that the pattern of expression ofsialic acids in certain cell types is controlled by specific expressionof sialyltransferases (Paulson, J. C. et al., 1989 J. Biol. Chem.264:10931-10934). The Siglec proteins may recognize not only theterminal sialic acids but also the context of these moieties based onpre-terminal sugars to which they are attached (Kelm, S., et al., 1996Glycoconj. J. 13:913-926).

[0009] Siglecs may mediate cell to cell adhesion by functioning assialic acid-dependent lectins with distinct specificities for the typeof sialic acid and its linkage to subterminal sugars (Kelm, S., 1994supra; Powell, L. D., et al., 1994 J. Biol. Chem. 269:10628-10636;Sjoberg, E., et al., 1994 J. Cell Biol. 126:549-562; Collins, B., E., etal., 1997 J. Biol. Chem. 272:1248-1255). For example, cells expressingSiglec-1 recognize the sequences Neu5Acα2,3Galβ1,3GalNAc andNeu5Acα2,3Galβ1,3(4)GlcNAc on glycoproteins and glycolipids (Kelm, S.,et al., 1994 Curr. Biol. 4:965:72; Crocker, P. R., et al., 1991 EMBO J.10:1661).

[0010] Siglecs are also postulated to be involved in cis-interaction inwhich a Siglec protein recognizes glycoconjugates on the same cell. Suchcis-interaction may regulate intercellular adhesion for CD22(Braesch-Andersen, S. and Stamenkovic, I. 1994 J. Biol. Chem.269:11783-11786; Hanasaki, K., et al., 1995 J. Biol. Chem.270:7533-7542), CD33 (Freeman, S. D., et al., 1995 supra), and MAG (asdiscussed in Freeman, S. D., et al., 1995 supra).

[0011] The amino acid sequences of the cytoplasmic tails of severalSiglec proteins strongly suggest that they participate in intracellularsignaling. For example, Siglec-2 has 6 tyrosines in the cytoplasmicdomain, two of which reside within ITAM (Immunotyrosine-based activationmotifs) motifs which mediate activation, and four within ITIM(Immunotyrosine-based inhibition motifs) motifs which mediate inhibition(Taylor, V. et al., 1999 J. Biol. Chem. 274:11505-11512).Phosphorylation of the ITAM motif tyrosines would allow recruitment ofSrc, whereas phosphorylation of ITIM motif tyrosines would allowrecruitment of SHP-1 and SHP-2. Siglec-3 contains two ITIMs that recruitSHP-1 and SHP-2 upon phosphorylation (Taylor, V. et al., 1999 supra).Siglec-6 also has putative SLAM-like signaling motifs in the cytoplasmictail; SLAM is an acronym for Signaling Lymphocyte Activation Molecule.(Patel, N. et al., 1999 J. Biol. Chem. 274:22729-22738).

[0012] Other biological activities of Siglecs have been postulated.There is mounting evidence that inflammatory cell infiltrates play asignificant role in driving the pathogenesis of asthma and otherallergic diseases by damaging tissue and releasing pro-inflammatoryagents. Activated eosinophils, neutrophils, macrophages, mast cells andlymphocytes increase in number at sites of inflammation and each arecapable of modifying the overall inflammatory response (Busse, W. W.1998 J. Allergy Clin. Immunol. 102:S17-22). Eosinophils are ofparticular interest in asthma and allergy due to their conspicuousappearance at the sites of allergen-driven inflammation (Kroegel, C. etal., 1994 Eur. Respir. J. 7:519-543; Haczku, A. 1998 Acta. Microbiol.Immunol. Hung. 45:19-29; Boyce, J. A. 1997 Allergy Asthma Proc.18:293-300). Through release of toxic granule proteins, pro-inflammatorylipid mediators and cytokines, eosinophils have been implicated as majorplayers in airway remodeling and hyperresponsiveness in asthma (Durham,S. R. 1998 Clin. Exp. Allergy 28 Suppl. 2:11-6).

[0013] CD33 (e.g., Siglec 3) is considered to be a member of the Siglecfamily based on its structural similarity with other Siglecs and itsability to bind to sialic acid. CD33 (Siglec-3; 67 kDa, Human sequencein EMBL/GENBANK M23197, Mouse sequence in EMBL/GENBANK S71345/S71403)was originally isolated from human myeloid cells (Andrews, R. G. et al1983 Blood 62:124; Griffin, J. D. et al 1984 Leuk Res. 8:521; Peiper, S.C. 1988 Blood 72:314-321; Peirelli, L et al., 1993 Br. J. Haematol.84:24;). Additional CD33 homologues have been identified, includingSAF-2 (European patent #EP 0 924 297 A1) and SAF-4 (published patentapplication No. WO9853840) and AF135027 (Genbank).

[0014] The sequences of the human (Simmons, D. L. and Seed, B. 1988 J.Immunol. 141: 2797) and murine (Tchilian, E. Z., et al., 1994 Blood83:3188) cDNA clones predict that CD33 encodes a Siglec having only twoC-set domains, making it the smallest of known Siglecs. CD33 binds toNeuAcα2,3Galβ1,3GalNAc in O-glycans and NeuAcα2,3Falβ1,3(4)GlcNAc inN-glycans (Freeman, S. et al., 1995 Blood 85:2005-2012). Cellsexpressing CD33 must be desialylated in order to bind to cells bearingthe appropriate sialoglycoconjugate, suggesting that inhibitorycis-interactions may regulate or block any adhesion function (Freeman,S. et al., 1995 supra). Additionally, CD33 has the conserved arginineresidue in the V-set domain.

[0015] CD33 is a clinically important diagnostic marker fordistinguishing myeloid from lymphoid leukemia (Griffin, J. D., et al.,Leuk. Res. 8:521; Matutes, E. et al., 1985 Haematol. Oncol. 3:179; Bain,B. J. ed 1990 in: Leukaemia Diagnosis. A Guide to the FABClassification, pp 61, London, UK, Gower Medical). CD33 expression hasbeen associated with myelomonocytic progenitors, monocytes andmacrophages, suggesting that it plays a role in regulating myeloid celldifferentiation (Peiper, S. C., et al., supra; Peirelli, L. et a.,supra; Andrews, R. G., et al., supra; Griffin, J. D., et al., supra;Nakamura, Y., et al., 1994 Blood 83:1442; Bernstein, I. D., et al., 1987J. Clin. Invest. 79:1153).

[0016] Sequences that are predicted to encode SIGLEC proteins that arestructurally similar to CD33 have been previously isolated andcharacterized. For example, sequences that are similar to CD33 include:CD33L1 and CD33L2 which are postulated to be related as a result ofdifferential-splicing and were isolated from a human placental cDNAlibrary (Takei, Y., et al., 1997 Cytogent. Cell. Genet. 78:295-300);Siglec-5 (Cornish, A. L., et al., 1998 Blood 92:2123-2132) which wasisolated from a human activated monocyte library (EST library #pHMQCD14)and is postulated to be expressed from the same gene that expressesOB-BP2 (Genbank Accession #: M23197) which is a leptin-binding protein,as they have nearly identical sequences; and Siglec-6 (OB-BP1) which wasisolated from the TF-1 human erythroleukemic cell line (Patel, N., etal., 1999 J. Biol. Chem. 274:22729-22738). Sequences for Siglecs 7 and 8have also recently been described (Nicoll, G., et al., 1999 J. Biol.Chem. 274:34089-34095; Floyd, H., et al., 2000 J. Biol. Chem.275:861-866).

[0017] The present invention relates to the discovery of nucleotidesequences (e.g., Siglec-BMS-L3a, -L3b, -L3c, -L3d, -L4a, -L5a, and -L5b)and novel Siglec proteins encoded by them having structural homology toCD33/Siglec 3.

SUMMARY OF THE INVENTION

[0018] The invention provides isolated nucleic acid molecules encodingthe SIGLEC-BMS proteins of the invention, and methods for uses thereof.For example, the nucleotide sequences of the invention include:Siglec-BMS-L3a, -L3b, -L3c, -L3d, -L4a, -L5a, L5b, and -L3-995-2 asshown in FIGS. 2A, 3A, 4A, 5A, 7A, 8A, 9A, and 6A respectively.

[0019] The invention further provides SIGLEC-BMS protein molecules.Specific embodiments of SIGLEC-BMS proteins of the invention include:SIGLEC-BMS-L3a, -L3b, -L3c, -L3d, -L4a, -L5a, -L5b, and -L3-995-2 asshown in FIGS. 2B, 3B, 4B, 5B, 7B, 8B, 9B, and 6B respectively.

[0020] The nucleic acid molecules of the invention include portions ofthe Siglec-BMS sequences, such as oligonucleotides, or fragmentsthereof. The nucleic acid molecules of the invention also includepeptide nucleic acids (PNA), and antisense molecules that react with thenucleic acid molecules of the invention.

[0021] The present invention also encompasses various nucleotidesequences that represent different forms of the Siglec-BMS genes andtranscripts, such as different allelic forms, polymorphic forms,alternative precursor transcripts, mature transcripts, anddifferentially-spliced transcripts. Additionally, recombinant nucleicacid molecules that are codon usage variants of the Siglec-BMS sequencesare provided.

[0022] The present invention includes the polynucleotides encodingSiglec-BMS in recombinant expression vectors and host-vector systemsthat include the expression vectors. One embodiment provides varioushost cells introduced with recombinant vectors that include theSiglec-BMS sequences of the invention.

[0023] The present invention provides methods for using isolated andsubstantially purified Siglec-BMS nucleotide sequences as nucleic acidprobes and primers, for using SIGLEC-BMS polypeptides as antigens forthe production of anti-SIGLEC-BMS antibodies, and for using SIGLEC-BMSpolypeptides for obtaining and detecting SIGLEC-BMS ligands. TheSiglec-BMS probes and primers, and the anti-SIGLEC-BMS antibodies areuseful in diagnostic assays and kits for the detection of naturallyoccurring Siglec-BMS nucleotide sequences and SIGLEC-BMS proteinsequences present in biological samples.

[0024] The invention also relates to antisense molecules capable ofreacting with the Siglec-BMS nucleotide sequences of the invention,thereby disrupting expression of genomic sequences.

[0025] The invention also relates to therapeutic agents includingagonists, antibodies, antagonists or inhibitors of the activity ofSIGLEC-BMS proteins. These compositions are useful for the prevention ortreatment of conditions associated with the presence or the deficiencyof SIGLEC-BMS proteins.

[0026] The present invention further provides pharmaceuticalcompositions for treating immune system diseases, such as asthma,leukemia, or other allergic or inflammatory diseases, comprising atleast one SIGLEC-BMS protein and a pharmaceutically acceptable carrier.The present invention further provides pharmaceutical compositionscomprising an antibody or antibody fragment thereof, that recognizes atleast one SIGLEC-BMS protein, in an acceptable carrier.

[0027] Kits comprising pharmaceutical compositions therapeutic forimmune system diseases are also encompassed by the invention. In oneembodiment, a kit comprising one or more of the pharmaceuticalcompositions of the invention is used to treat an immune system disease,e.g. asthma, leukemia, or other allergic or inflammatory diseases.

BRIEF DESCRIPTION OF THE FIGURES

[0028]FIG. 1: Schematic representation of the predicted structures ofthe SIGLEC family of proteins.

[0029]FIG. 2: A) The nucleotide sequence of Siglec-BMS-L3a (SEQ IDNO.:1); B) the predicted amino acid sequence of SIGLEC-BMS-L3a (SEQ IDNO.:8), as described in Example 1, infra.

[0030]FIG. 3: A) The nucleotide sequence of Siglec-BMS-L3b (SEQ IDNO.:2); B) the predicted amino acid sequence of SIGLEC-BMS-L3b (SEQ IDNO.:9), as described in Example 1, infra.

[0031]FIG. 4: A) The nucleotide sequence of Siglec-BMS-L3c (SEQ IDNO.:3); B) the predicted amino acid sequence of SIGLEC-BMS-L3c (SEQ IDNO.:10), as described in Example 1, infra.

[0032]FIG. 5: A) The nucleotide sequence of Siglec-BMS-L3d (SEQ IDNO.:4); B) the predicted amino acid sequence of SIGLEC-BMS-L3d (SEQ IDNO.: 11), as described in Example 1, infra.

[0033]FIG. 6: A) The nucleotide sequence of Siglec-BMS-L3-995-2 (SEQ IDNO.:27), the ATG start codon and the splicing locations are shaded, theopen boxed region represents the transmembrane domain; B) the predictedamino acid sequence of SIGLEC-BMS-L3-995-2 (SEQ ID NO.:28), as describedin Example 14, infra.

[0034]FIG. 7: A) The nucleotide sequence of Siglec-BMS-L4a (SEQ IDNO.:5); B) the predicted amino acid sequence of SIGLEC-BMS-L4a (SEQ IDNO.:12), as described in Example 1, infra.

[0035]FIG. 8: A) The nucleotide sequence of Siglec-BMS-L5a (SEQ IDNO.:6); B) the predicted amino acid sequence of SIGLEC-BMS-L5a (SEQ IDNO.:13), as described in Example 1, infra.

[0036]FIG. 9: A) The nucleotide sequence of Siglec-BMS-L5b (SEQ IDNO.:7); B) the predicted amino acid sequence of SIGLEC-BMS-L5b (SEQ IDNO.:14), as described in Example 1, infra.

[0037]FIG. 10: A) A Northern blot analysis showing the distribution ofSiglec-BMS-L3 transcripts in human tissue; B) a schematic map showingthe location of the probe sequences, as described in Example 2, infra.

[0038]FIG. 11: A) A table showing the results of an RT-PCR analysisshowing the distribution of Siglec-BMS-L3 transcripts in human tissue;B) a schematic map showing the location of the primers/PCR products, asdescribed in Example 3, infra.

[0039]FIG. 12: A) Histograms showing the distribution of Siglec-BMS-L3transcripts in human tissue and cell lines, as detected by quantitativeRT-PCR analysis; B) a quantitative RT-PCR analysis showing expressionlevels of Siglec-BMS-L3 transcripts in purified human white blood cellsfrom two individual human subjects; C) a schematic map showing thelocation of the primers/PCR products, as described in Example 4, infra.

[0040]FIG. 13: A graph showing the results of a binding assay in whichimmobilized SIGLEC-BMSL3-hIg fusion protein (e.g., extracellular domainof SIGLEC-BMS-L3) binds to various blood cell populations or cell lines,as described in Example 8, infra.

[0041]FIG. 14: A graph showing the results of a binding assay in whichCOS7 cells, expressing full-length SIGLEC-BMS-L3 protein, bind tovarious blood cell populations or cell lines, as described in Example 9,infra.

[0042]FIG. 15: A schematic representation of the various GST fusionproteins comprising the cytoplasmic tail of wild-type and mutatedSIGLEC-BMS-L3 protein, including L3cyto-wt, L3cyto-Y641F, L3cyto-Y667F,L3cyto-Y691F, and L3cyto-Y641 alone. Also depicted are hIg (humanimmunoglobulin) fusion proteins comprising the non-spliced (995-2,SIGLEC-BMSL3 hIg) and spliced (526604, SIGLEC-BMSL3a hIg) extracellulardomains of SIGLEC-BMSL3 protein, as described in Example 10, infra.

[0043]FIG. 16: Graphs showing the results of kinase assays involvingvarious substrates including GST fusion proteins comprising thecytoplasmic tail of wild-type and mutated SIGLEC-BMS-L3 protein reactedwith various tyrosine kinases, as described in Example 12, infra: A) lckkinase; B) ZAP70 kinase; C) emt kinase; and D) JAK3 kinase. E) A graphshowing the results of kinase assays involving a GST fusion proteinsubstrate, comprising the cytoplasmic tail of wild-type SIGLEC-BMS-L3protein, and various tyrosine kinases including lck, ZAP70, emt, andJAK3. F) A graph showing the results of kinase assays involving LATsubstrate and various tyrosine kinases including lck, ZAP70, emt, andJAK3. G) A graph showing results of tyrosine phosphorylation of GSTfusion proteins comprising the cytoplasmic tail of wild-type and variousY→F mutants with a tyrosine kinase mix.

[0044]FIG. 17: A) Results of immunoprecipitation experimentsdemonstrating that SHP-1 and SHP-2 associate with the phosphorylatedSIGLEC-BMSL3 cytoplasmic tail, as described in Example 13, infra. B)Depicts binding of SHP-1 and SHP-2 to Y667 ITIM by ELISA, as describedin Example 13.

[0045]FIG. 18: Depicts the nucleotide and amino acid sequences of thecytoplasmic tail domain of Siglec-BMS-L3a fused to a GST protein asdescribed in Example 10, infra. A) The nucleotide sequence of L3cyto-wt(SEQ ID NO: 17); B) the amino acid sequence of L3cyto-wt (SEQ ID NO:22).

[0046]FIG. 19: Depicts the nucleotide and amino acid sequences of thecytoplasmic tail domain of Siglec-BMS-L3a fused to a GST protein, asdescribed in Example 10, infra. A) The nucleotide sequence ofL3cyto-Y641F (SEQ ID NO:18); B) the amino acid sequence of L3cyto-Y641F(SEQ ID NO:23).

[0047]FIG. 20: Depicts the nucleotide and amino acid sequences of thecytoplasmic tail domain of Siglec-BMS-L3a fused to a GST protein, asdescribed in Example 10, infra. A) The nucleotide sequence ofL3cyto-Y667F (SEQ ID NO:19); B) the amino acid sequence of L3cyto-Y667F(SEQ ID NO:24).

[0048]FIG. 21: Depicts the nucleotide and amino acid sequences of thecytoplasmic tail domain of Siglec-BMS-L3a fused to a GST protein, asdescribed in Example 10, infra. A) The nucleotide sequence ofL3cyto-Y691F (SEQ ID NO:20); B) the amino acid sequence of L3cyto-Y691F(SEQ ID NO:25).

[0049]FIG. 22: Depicts the nucleotide and amino acid sequences of thecytoplasmic tail domain of Siglec-BMS-L3a fused to a GST protein, asdescribed in Example 10, infra. A) The nucleotide sequence ofL3cyto-Y641 alone (SEQ ID NO:21); B) the amino acid sequence ofL3cyto-Y641 alone (SEQ ID NO:26).

[0050]FIG. 23: Depicts the nucleotide and amino acid sequences of theextracellular domain of Siglec-BMS-L3a fused to a human immunoglobulinprotein (hIg), as described in Example 11, infra. A) The nucleotidesequence of Siglec-BMS-L3a hIg (SEQ ID NO:31); B) the amino acidsequence of SIGLEC-BMS-L3a hIg (SEQ ID NO:32).

[0051]FIG. 24: Depicts the nucleotide and amino acid sequence of theextracellular domain of Siglec-BMS-L3 fused to a human immunoglobulinprotein, as described in Example 11, infra. A) The nucleotide sequenceof Siglec-BMS-L3 hIg (SEQ ID NO:29), B) the amino acid sequence ofSIGLEC-BMS-L3 hIg (SEQ ID NO:30).

[0052]FIG. 25: Depicts the 697 amino acid sequence for Siglec-10,predicted based on the longest open reading frame (SEQ ID NO:15). Thetwo spliced regions are indicated in gray, the cryptic splice acceptorsite is underlined, the transmembrane domain is bolded and amino acidsin the ITEM motifs in the cytoplasmic domain are boxed. The intron/exonboundaries are indicated with arrow and the domain numbers reflect thefive Ig-like domains.

[0053]FIG. 26: Depicts the binding of polyacrylamide glycoconjugate toSiglec-10 as described in Example 15. Results shown are a mean +/−SD of2 experiments, n=4-6 treatment/experiment.

[0054]FIG. 27: Depicts the Western blot of cell lysate probed withanti-Siglec-10 monoclonal antibody as described in Example 16.

[0055]FIG. 28: Depicts the results of in situ hybridization (ISH)detailing the distribution of Siglec-10 positive hybridization signalsin non-human primate and human tissues as described in Example 17.

[0056] A) NHP spleen (Panels A, C, E); human spleen (Panels B, D, E)

[0057] B) NHP jejunum (Panels A, C, E); human liver (Panels B, D, E)

[0058] C) NHP Colon (Panels A-G)

[0059] D) NHP lymph nodes (Panels A, C, E); human lymph node (Panels B,D, E)

[0060] E) Human asthma lung (Panels A, C, E)

[0061] F) NHP lung (Panels A, B, D, E, G, H); human lung (Panels C, F,I)

DETAILED DESCRIPTION OF THE INVENTION

[0062] In order that the invention herein described may be more fullyunderstood, the following description is set forth.

[0063] The term “Siglec-BMS” as used herein refers to a protein familyof sialic acid-binding Ig-like lectins sharing structural similarityincluding at least one Ig-like domain, a transmembrane domain, and acytoplasmic tail. Typically, the Ig-like domain is extracellular andcomprises an Ig(V) domain and an Ig(C) domain. Examples of SIGLEC-BMSproteins include, but are not limited to, L3a, L3b, L3c, L3d, L3-995-2,L4a, L5a, and L5b.

[0064] The term “Siglec-10” as used herein refers to a protein family ofsialic acid -binding Ig-like lectins that shares structural similarityto CD33-related Siglecs, including multiple Ig-like domains, atransmembrane domain, and a cytoplasmic tail containing twoITIM-signaling motifs. The full length Siglec-10 protein comprises fiveIg-like domains (Ig-DI, Ig-D2, Ig-D3, Ig-D4, and Ig-D5), and isdesignated SIGLEC-BMS-L3 in this application. The full length Siglec-10protein is also termed as SIGLEC-BMS-L3-995-2. The terms Siglec-10,SIGLEC-BMS-L3, and SIGLEC-BMS-L3-995-2 are used interchangeably in thisapplication.

[0065] The term “isolated” as used herein means a specific nucleic acidor polypeptide, or a fragment thereof, in which contaminants (i.e.substances that differ from the specific nucleic acid or polypeptidemolecule) have been separated from the specific nucleic acid orpolypeptide.

[0066] The term “purified” as used herein means a specific isolatednucleic acid or polypeptide, or a fragment thereof, in whichsubstantially all contaminants (i.e. substances that differ from thespecific nucleic acid or polypeptide molecule) have been separated fromthe specific nucleic acid or polypeptide.

[0067] As used herein, a first nucleotide or amino acid sequence is saidto have sequence “identity” to a second reference nucleotide or aminoacid sequence, respectively, when a comparison of the first and thereference sequences shows that they are exactly alike.

[0068] As used herein, a first nucleotide or amino acid sequence is saidto be “similar” to a second reference sequence when a comparison of thetwo sequences shows that they have few sequence differences (i.e., thefirst and second sequences are nearly identical). For example, twosequences are considered to be similar to each other when the percentageof nucleotides or amino acids that differ between the two sequences maybe between about 60% to 99.99%.

[0069] The term “complementary” as used herein refers to nucleic acidmolecules having purine and pyrimidine nucleotides which have thecapacity to associate through hydrogen bonding to form double strandednucleic acid molecules. The following base pairs are related bycomplementarity: guanine and cytosine; adenine and thymine; and adenineand uracil. Complementary applies to all base pairs comprising twosingle-stranded nucleic acid molecules, or to all base pairs comprisinga single-stranded nucleic acid molecule folded upon itself.

[0070] The term “fragment” of a SIGLEC-BMS-encoding nucleic acidmolecule refers to a portion of a nucleotide sequence which encodes apolypeptide having the biological activity of a SIGLEC-BMS protein. Afragment of a Siglec-BMS molecule, is therefore, a nucleotide sequencehaving fewer nucleotides than the nucleotide sequence encoding theentire amino acid sequence of a SIGLEC-BMS protein, and which encodes apeptide having the biological activity of a SIGLEC-BMS protein

[0071] The term “fragment” of a SIGLEC-BMS polypeptide molecule refersto a portion of a polypeptide having the biological activity of aSIGLEC-BMS polypeptide.

[0072] The term “biological activity” of a SIGELC-BMS protein as usedherein means that the protein functions as a cell adhesion moleculeand/or the protein elicits the generation of an anti-SIGLEC-BMSantibody, where the SIGLEC-BMS protein binds with an anti-SIGLEC-BMSantibody.

[0073] The term “heterologous” as used herein refers to a non-SIGLEC-BMSprotein or a fragment thereof. The heterologous molecule is fused (e.g.,linked or joined) to a SIGLEC-BMS protein to facilitate isolation and/orpurification of expressed SIGLEC-BMS gene product. Examples ofheterologous molecules include, but are not limited to, humanimmunoglobulin constant region, a His-tag sequence, or a glutathioneS-transferase (GST) sequence.

[0074] Molecules of the Invention

[0075] In its various aspects, as described in detail below, the presentinvention provides proteins, antibodies, nucleic acid molecules,recombinant DNA molecules, transformed host cells, generation methods,assays, therapeutic plus diagnostic methods and pharmaceutical,therapeutic or diagnostic compositions, all involving a Siglec-BMSprotein or nucleic acids encoding them.

[0076] For the sake of convenience, the nucleotide sequences ofSiglec-BMS (e.g., -L3a, -L3b, -L3c, -L3d, -L3-995-2,-L4a, -L5a, and-L5b) will be collectively referred to as “Siglec-BMS” or “Siglecnucleotide sequences of the invention”. Additionally, the proteinsencoded by the Siglec-BMS nucleotide sequences include “SIGLEC-BMS-L3a,-L3b, -L3c, -L3d, -L3-995-2,-L4a, -L5a, and -L5b proteins” andcollectively referred to as “SIGLEC-BMS proteins” or “SIGLEC proteins ofthe invention” or “proteins of the invention”.

[0077] Nucleic Acid Molecules of the Invention

[0078] Nucleic Acid Molecules Encoding SIGLEC-BMS Proteins

[0079] The present invention discloses the discovery of nucleic acidmolecules, herein termed Siglec-BMS nucleotide sequences, that encodenovel polypeptides having similar structural features shared by proteinsin the Siglec subgroup. Structural features shared by the Siglecsubgroup include an Ig-like domain which is extracellular and comprisesa C-set domain and a V-set domain having an unusual intrasheet disulfidebridge between the B and E strands (A. F. Williams and A. N. Barclay1988 Annu. Rev. Immunol. 6:381-405; A. F. Williams, et al. 1989 ColdSpring Harbor Symp. Quant. Biol. 54:637-647; L. Pedraza, et al. 1990 J.Cell biol. 111:2651-2661). The nucleotide sequences of Siglec-BMS encodepolypeptides each can have two (e.g., -L4,-L5a, and -L5b) to three(e.g., -L3a, -L3b, -L3c, and -L3d) C-set domains.

[0080] In particular embodiments, novel nucleotide sequences aredesignated L3a, L3b, L3c, L3d, L4, L5a, L5b, and L3-995-2, as shownshown in FIGS. 2A, 3A, 4A, 5A, 7A, 8A, 9A, and 6A respectively (SEQ IDNOS.:1-7 and 27). These nucleotide sequences encode SIGLEC-BMS proteinsand/or fragments thereof, where the encoded proteins exhibit abiological activity, for example, functioning as a cell adhesionmolecule. For example, an isolated Siglec nucleic acid encoding L3a isshown in FIG. 2A beginning at codon GGC at position +12 and ending atcodon CCA at position +1760. An isolated Siglec nucleic acid encodingL3b is shown in FIG. 3A beginning at codon GAT at position +3 and endingat codon CAA at position +1868. An isolated Siglec nucleic acid encodingL3c is shown in FIG. 4A beginning at codon GGA at position +12 andending at codon CAA at position +1736. An isolated Siglec nucleic acidencoding L3d is shown in FIG. 5A beginning at codon CCC at position +2and ending at codon ATG at position +1291. An isolated Siglec nucleicacid encoding L3 is shown in FIG. 6A beginning at codon ATG at position+1 and ending at codon CAA at position +2091. An isolated Siglec nucleicacid encoding L4a is shown in FIG. 7A beginning at codon CTG at position+1 and ending at codon GGC at position +1398. An isolated Siglec nucleicacid encoding L5a is shown in FIG. 8A beginning at codon ATG at position+43 and ending at codon AGA at position +1431. An isolated Siglecnucleic acid encoding L5b is shown in FIG. 9A beginning at codon ATG atposition +57 and ending at codon AGT at position +914.

[0081] Siglec-BMS-L3, Siglec-BMS-L4 (also referred to herein as L4a),Siglec-BMS-L5a and Siglec-BMS-L5b were collectively deposited on Aug.10, 2000 with the American Type Culture Collection (ATCC), 10801University Blvd., Manassas, Va. 20110-2209 under the provisions of theBudapest Treaty, and has been accorded ATCC accession number PTA-2343.The nucleic acid sequences of each of Siglec-BMS-L3, Siglec-BMS-L4 (alsoreferred to herein as L4a), Siglec-BMS-L5a and Siglec-BMS-L5b areprovided in FIGS. 6A, 7A, 8A and 9A respectively. These nucleic acidsequences can be easily separated from the collective deposit bystandard separation techniques such as hybridization to specific probesor restriction analyses (Maniatis, T., et al., 1989 Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.).

[0082] In accordance with the practice of the invention, the nucleotidesequences of the invention may be isolated full-length or partial cDNAmolecules or oligomers of the Siglec-BMS sequences. A Siglec-BMSnucleotide sequence can encode all or portions of the signal peptideregion, the extracellular domain, the transmembrane domain, and/or theintracellular domain of a SIGLEC-BMS protein.

[0083] Isolated Siglec-BMS Sequences

[0084] The nucleic acid molecules of the invention are preferably inisolated form, where the nucleic acid molecules are substantiallyseparated from contaminant nucleic acid molecules having sequences otherthan Siglec-BMS sequences. A skilled artisan can readily employ nucleicacid isolation procedures to obtain isolated Siglec-BMS sequences, seefor example Sambrook et al., Molecular Cloning (1989). The presentinvention also provides for isolated Siglec-BMS sequences generated byrecombinant DNA technology or chemical synthesis methods. The presentinvention also provides nucleotide sequences isolated from variousmammalian species including, bovine, ovine, porcine, murine, equine, andpreferably, human species.

[0085] The isolated nucleic acid molecules include DNA, RNA, DNA/RNAhybrids, and related molecules, nucleic acid molecules complementary tothe SIGLEC-BMS encoding sequences or a portion thereof, and those whichhybridize to the nucleic acid sequences that encode the SIGLEC-BMSproteins. The preferred nucleic acid molecules have nucleotide sequencesidentical to or nearly identical (e.g., similar) to the nucleotidesequences disclosed herein. Specifically contemplated are genomic DNA,cDNA, ribozymes, and antisense molecules.

[0086] Identical and Variant Siglec-BMS Sequences

[0087] The present invention provides isolated nucleic acid moleculeshaving a polynucleotide sequence identical or similar to the Siglec-BMSsequences disclosed herein. Accordingly, the polynucleotide sequencesmay be identical to a particular Siglec-BMS sequence, as described inSEQ ID NOS.:1-7 or 27. Alternatively, the polynucleotide sequences maybe similar to the disclosed sequences.

[0088] One embodiment of the invention provides nucleic acid moleculesthat exhibit sequence identity or similarity with the Siglec-BMSnucleotide sequences, such as molecules that have at least 60% to 99.9%sequence similarity and up to 100% sequence identity with the sequencesof the invention as shown in FIGS. 2A, 3A, 4A, 5A, 7A, 8A, 9A and 6A(SEQ ID NOS.:1-7, or 27). A preferred embodiment provides nucleic acidmolecules that exhibit between about 75% to 99.9% sequence similarity, amore preferred embodiment provides molecules that have between about 86%to 99.9% sequence similarity, and the most preferred embodiment providesmolecules that have 100% sequence identity with the Siglec-BMS sequencesof the invention (e.g., SEQ ID NOS.:1-7, or 27).

[0089] Differentially Spliced Siglec-BMS Sequences

[0090] The nucleic acid molecules of the present invention comprisenucleic acid sequences corresponding to differentially splicedtranscripts of Siglec-BMS. In general, a differentially-splicedtranscript is a mature RNA transcript that can be generated in a cell bythe following steps: (1) the cell transcribes precursor RNA transcriptsfrom an intron-containing gene, where the precursor RNA transcriptsinclude all the intron sequences; (2) the cell splices out differentintrons from different precursor transcripts, resulting in aheterogeneous population of mature RNA transcripts each having differentintrons; (3) the cell translates some or all of thedifferentially-spliced transcripts to generate a heterogeneouspopulation of proteins which are encoded by the same intron-containinggene sequence. Thus, a cell may produce a heterogeneous population ofSiglec-BMS RNA transcripts that are related to each other as a result ofdifferential splicing of a common precursor transcript. Furthermore, theSIGLEC-BMS proteins that are translated from the differentially splicedtranscripts may have different biological activities.

[0091] For example, the polynucleotide sequences of the presentinvention include introns and can encode three different classes ofSIGLEC-BMS proteins: (1) the nucleotide sequences described in FIGS. 2A,3A, 4A, and 5A (SEQ ID NOS.: 1-4) which represent cDNA clones that arerelated to each other and correspond to differentially splicedtranscripts of Siglec-BMS-L3a, -L3b, -L3c, and -L3d, which encodeSIGLEC-BMS proteins -L3 a, -L3b, -L3c, and -L3d respectively (e.g.,FIGS. 2b, 3 b, 4B, and 5B respectively; SEQ ID NOS.: 8, 9, 10 and 11,respectively); (2) the nucleotide sequence described in FIG. 7A (SEQ IDNO.: 5) which represents a cDNA clone that corresponds to adifferentially spliced transcript of Siglec-BMS-L4a which encodesSIGLEC-BMS-4a protein (FIG. 7B; SEQ ID NOS.: 12); (3) the nucleotidesequences described by FIGS. 8A and 9A (SEQ ID NOS.: 6-7) whichrepresent cDNA clones that are related to each other and correspond todifferentially spliced transcripts of Siglec-BMS-L5a, and -L5b, whichencode SIGLEC-BMS-5a and -5b proteins, respectively (e.g., FIGS. 8B and9b; SEQ ID NOS.:13 and 14, respectively). The invention also providesnucleic acid molecules having the nucleotide sequence ofSiglec-BMS-L3-995-2 (FIG. 6A; SEQ ID NO.:27), which represents a hybridconstruct of full-length Siglec-BMS-L3 cDNA (Example 14, infra).

[0092] Complementary Sequences

[0093] The invention also provides nucleic acid molecules that arecomplementary to the sequences as described in FIGS. 2A, 3A, 4A, 5A, 7A,8A, 9A, and 6A (SEQ ID NO: 1-7, and 27) (preferably, the codingsequences excluding the vector sequences therein). Complementarity maybe full or partial. When it is fully complementary that meanscompementarity to the entire sequence as described in SEQ ID NO:1-7, and27. When it is partially complementary that means complementarity toonly portions of sequences as described in SEQ ID NO: 1-7, and 27.

[0094] Nucleotide Sequences Which Hybridize to Siglec-BMS Sequences

[0095] The present invention further provides nucleotide sequences thatselectively hybridize to Siglec-BMS nucleotide sequences (e.g., SEQ IDNO.: 1-7, or 27) under high stringency hybridization conditions.Typically, hybridization under standard high stringency conditions willoccur between two complementary nucleic acid molecules that differ insequence complementarity by about 70% to about 100%. It is readilyapparent to one skilled in the art that the high stringencyhybridization between nucleic acid molecules depends upon, for example,the degree of identity, the stringency of hybridization, and the lengthof hybridizing strands. The methods and formulas for conducting highstringency hybridizations are well known in the art, and can be foundin, for example, Sambrook, et al., Molecular Cloning (1989).

[0096] In general, stringent hybridization conditions are those that:(1) employ low ionic strength and high temperature for washing, forexample, 0.015M NaCl/0.0015M sodium titrate/0.1% SDS at 50 degrees C.;or (2) employ during hybridization a denaturing agent such as formamide,for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM NaCl, 75 mM sodium citrate at 42 degrees C.

[0097] Another example of stringent conditions include the use of 50%formamide, 5× SSC (0.75M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt's solution,sonicated salmon sperm DNA (50 mg/ml), 0.1% SDS, and 10% dextran sulfateat 42 degrees C., with washes at 42 degrees C. in 0.2× SSC and 0.1% SDS.A skilled artisan can readily determine and vary the stringencyconditions appropriately to obtain a clear and detectable hybridizationsignal.

[0098] Fragments of Siglec-BMS Sequences

[0099] The invention further provides nucleic acid molecules havingfragments of the Siglec-BMS sequences of the invention, such as aportion of the Siglec-BMS sequences disclosed herein (as shown in SEQ IDNO.:1-7, 15, and 27). The size of the fragment will be determined by itsintended use. For example, if the fragment is chosen to encode aSIGLEC-BMS extracellular domain, then the skilled artisan shall selectthe polynucleotide fragment that is large enough to encode thisdomain(s). If the fragment is to be used as a nucleic acid probe or PCRprimer, then the fragment length is chosen to obtain a relatively smallnumber of false positives during a probing or priming procedure.Alternatively, a fragment of the Siglec-BMS sequence may be used toconstruct a recombinant fusion gene having a Siglec-BMS sequence fusedto a non-Siglec-BMS sequence, such as a human immunoglobulin or a GSTsequence.

[0100] The nucleic acid molecules, fragments thereof, and probes andprimers of the present invention are useful for a variety of molecularbiology techniques including, for example, hybridization screens oflibraries, or detection and quantification of mRNA transcripts as ameans for analysis of gene transcription and/or expression. Preferably,the probes and primers are DNA. A probe or primer length of at least 15base pairs is suggested by theoretical and practical considerations(Wallace, B. and Miyada, G. 1987 in: “Oligonucleotide Probes for theScreening of Recombinant DNA Libraries” in: Methods in Enzymology,152:432-442, Academic Press).

[0101] Fragments of Siglec-BMS nucleotide sequences that areparticularly useful as selective hybridization probes or PCR primers canbe readily identified from the Siglec-BMS nucleotide sequences, usingart-known methods. For example, sets of PCR primers that detect theportion of Siglec-BMS transcripts that encode the extracellular domainof a SIGLEC protein can be made by the PCR method described in U.S. Pat.No. 4,965,188. The probes and primers of this invention can be preparedby methods well known to those skilled in the art (Sambrook, et al.supra). In a preferred embodiment the probes and primers are synthesizedby chemical synthesis methods (ed: Gait, M. J. 1984 OligonucleotideSynthesis, IRL Press, Oxford, England).

[0102] One embodiment of the present invention provides nucleic acidprimers that are complementary to Siglec-BMS sequences, which allow thespecific amplification of nucleic acid molecules of the invention or ofany specific portions thereof. Another embodiment provides nucleic acidprobes that are complementary for selectively or specificallyhybridizing to the Siglec-BMS sequences or to any portion thereof, e.g.,a all or portion of the extracellular domain.

[0103] Fusion Genes

[0104] The present invention provides fusion genes, which include aSiglec-BMS sequence fused (e.g., linked or joined) to a non-Siglec-BMSsequence such as, for example, a HIS-tag sequence, to facilitateisolation and/or purification of the expressed SIGLEC-BMS gene product(Kroll, D. J., et al., 1993 DNA Cell Biol 12:441-53), or a GST, or ahuman immunoglobulin sequence. The preferred fusion gene comprises aSiglec-BMS sequence operatively linked to a non-Siglec-BMS sequence,such as, for example a Siglec-BMS sequence fused in-frame with anon-Siglec-BMS sequence.

[0105] Alternatively, the fusion genes of the invention include aSiglec-BMS sequence fused to a Siglec-BMS sequence isolated from adifferent mammalian source. For example, the human Siglec-BMS sequences,disclosed herein, can be fused to a Siglec-BMS sequence isolated from adifferent human or a different mammalian species.

[0106] Codon Usage Variants Encoding SIGLEC-BMS Proteins

[0107] The present invention provides isolated codon-usage variants thatdiffer from the disclosed Siglec-BMS nucleotide sequences, yet do notalter the predicted SIGLEC-BMS polypeptide sequence or biologicalactivity. For example, a number of amino acids are designated by morethan one triplet. Codons that specify the same amino acid, or synonymsmay occur due to degeneracy in the genetic code. Examples includenucleotide codons CGT, CGG, CGC, and CGA encoding the amino acid,arginine (R); or codons GAT, and GAC encoding the amino acid, asparticacid (D). Thus, a protein can be encoded by one or more nucleic acidmolecules that differ in their specific nucleotide sequence, but stillencode protein molecules having identical sequences. The amino acidcoding sequence is as follows: One Letter Amino Acid Symbol SymbolCodons Alanine Ala A GCU, GCC, GCA, GCG Cysteine Cys C UGU, UGC AsparticAcid Asp D GAU, GAC Glutamic Acid Glu E GAA, GAG Phenylalanine Phe FUUU, UUC Glycine Gly G GGU, GGC, GGA, GGG Histidine His H CAU, CACIsoleucine Ile I AUU, AUC, AUA Lysine Lys K AAA, AAG Leucine Leu L UUA,UUG, CUU, CUC, CUA, CUG Methionine Met M AUG Asparagine Asn N AAU, AACProline Pro P CCU, CCC, CCA, CCG Glutamine Gln Q CAA, CAG Arginine Arg RCGU, CGC, CGA, CGG, AGA, AGG Serine Ser S UCU, UCC, UCA, UCG, AGU, AGCThreonine Thr T ACU, ACC, ACA, ACG Valine Val V GUU, GUC, GUA, GUGTryptophan Trp W UGG Tyrosine Tyr Y UAU, UAC

[0108] The codon-usage variants may be generated by recombinant DNAtechnology. Codons may be selected to optimize the level of productionof the Siglec-BMS transcript or SIGLEC-BMS polypeptide in a particularprokaryotic or eukaryotic expression host, in accordance with thefrequency of codon utilized by the host cell. Alternative reasons foraltering the nucleotide sequence encoding a SIGLEC-BMS polypeptideinclude the production of RNA transcripts having more desirableproperties, such as an extended half-life or increased stability. Amultitude of variant Siglec-BMS nucleotide sequences that encode therespective SIGLEC-BMS polypeptide may be isolated, as a result of thedegeneracy of the genetic code. Accordingly, the present inventionprovides selecting every possible triplet codon to generate everypossible combination of nucleotide sequences that encode the disclosedSIGLEC-BMS polypeptides, or that encode polypeptides having thebiological activity of the SIGLEC-BMS polypeptides. This particularembodiment provides isolated nucleotide sequences that vary from thesequences as described in SEQ ID NOS.: 1-7, or 27, such that eachvariant nucleotide sequence encodes a polypeptide having sequenceidentity with the amino acid sequences, as described in FIGS. 2B, 3B,4B, 5B, 7B, 8B, 9B, or 6B (SEQ ID NOs.: 8-14, or 28), respectively.

[0109] Allelic Forms of Siglec-BMS Sequences

[0110] The present invention contemplates alternative allelic forms ofthe Siglec-BMS nucleotide sequences. These alternative allelic forms canbe isolated from different subjects of the same species.

[0111] Typically, isolated allelic forms of naturally-occurring genesequences include wild-type and mutant alleles. A wild-type Siglec-BMSgene sequence will encode a SIGLEC-BMS protein having normal SIGLEC-BMSbiological activity, such as, for example, function as a cell adhesionmolecule. A mutant Siglec-BMS gene sequence may encode a SIGLEC-BMSprotein having an activity not found in normal SIGLEC-BMS proteins, suchas, for example, not functioning as a cell adhesion molecule.Alternatively, a mutant Siglec-BMS gene sequence may encode a SIGLEC-BMSprotein having normal activity.

[0112] It will be appreciated by one skilled in the art that variationsin one or more nucleotides (up to about 3-4% of the nucleotides) of thenucleic acids encoding peptides having the activity of a SIGLEC-BMSmolecule may exist among individuals within a population due to naturalallelic variation. Any and all such nucleotide variations and resultingamino acid polymorpism are within the scope of the invention.

[0113] Polymorphic Forms of Siglec-BMS Sequences

[0114] The present invention provides nucleotide sequences of particularpolymorphic forms of Siglec-BMS, as described in SEQ ID NOS.: 1-7, or27. Typically, isolated polymorphic forms of naturally-occurring genesequences are isolated from different subjects of the same species. Thepolymorphic forms include sequences having one or more nucleotidesubstitutions that may or may not result in changes in the amino acidcodon sequence. These substitutions may result in a wild-type Siglec-BMSgene that encodes a protein having the biological activity of wild-typeSIGLEC-BMS proteins, or encodes a mutant polymorphic form of theSIGLEC-BMS protein having a different or null activity.

[0115] Derivative Nucleic Acid Molecules

[0116] The nucleic acid molecules of the invention also includederivative nucleic acid molecules which differ from DNA or RNAmolecules, and anti-sense molecules. Derivative molecules includepeptide nucleic acids (PNAs), and non-nucleic acid molecules includingphosphorothioate, phosphotriester, phosphoramidate, andmethylphosphonate molecules, that bind to single-stranded DNA or RNA ina base pair-dependent manner (Zamecnik, P. C., et al., 1978 Proc. Natl.Acad. Sci. 75:280284; Goodchild, P. C., et al., 1986 Proc. Natl. Acad.Sci. 83:4143-4146). Peptide nucleic acid molecules comprise a nucleicacid oligomer to which an amino acid residue, such as lysine, and anamino group have been added. These small molecules, also designatedanti-gene agents, stop transcript elongation by binding to theircomplementary (template) strand of nucleic acid (Nielsen, P. E., et al.,1993 Anticancer Drug Des 8:53-63). Reviews of methods for synthesis ofDNA, RNA, and their analogues can be found in: Oligonucleotides andAnalogues, eds. F. Eckstein, 1991, IRL Press, New York; OligonucleotideSynthesis, ed. M. J. Gait, 1984, IRL Press, Oxford, England.Additionally, methods for antisense RNA technology are described in U.S.Pat. Nos. 5,194,428 and 5,110,802. A skilled artisan can readily obtainthese classes of nucleic acid molecules using the herein describedSiglec-BMS polynucleotide sequences, see for example Innovative andPerspectives in Solid Phase Synthesis (1992) Egholm, et al. pp 325-328or U.S. Pat. No. 5,539,082.

[0117] RNA Encoding Siglec-BMS Polypeptides

[0118] The present invention provides nucleic acid molecules that encodeSIGLEC-BMS proteins. In particular, the RNA molecules of the inventionmay be isolated full-length or partial mRNA molecules or RNA oligomersthat encode the SIGLEC-BMS proteins. The RNA molecules of the inventionalso include antisense RNA molecules, peptide nucleic acids (PNAs), ornon-nucleic acid molecules such as phosphorothioate derivatives, thatspecifically bind in a base-dependent manner to the sense strand of DNAor RNA, having the Siglec-BMS sequences, in a base-pair manner. Askilled artisan can readily obtain these classes of nucleic acidmolecules using the Siglec-BMS sequences described herein.

[0119] Nucleic Acid Molecules Labeled With A Detectable Marker

[0120] Embodiments of the Siglec-BMS nucleic acid molecules of theinvention include DNA and RNA primers, which allow the specificamplification of Siglec-BMS sequences, or of any specific parts thereof,and probes that selectively or specifically hybridize to Siglec-BMSsequences or to any part thereof. The nucleic acid probes can be labeledwith a detectable marker. Examples of a detectable marker include, butare not limited to, a radioisotope, a fluorescent compound, abioluminescent compound, a chemiluminescent compound, a metal chelatoror an enzyme. Technologies for generating labeled DNA and RNA probes arewell known, see, for example, Sambrook et al., in Molecular Cloning(1989).

[0121] Proteins and Polypeptides of the Invention

[0122] The invention also provide novel SIGLEC-BMS proteins. Oneembodiment of a SIGLEC-BMS protein comprises an amino acid sequencebeginning with Ala141 and ending with Ser198 as shown in FIG. 6B (SEQ IDNO:28). Another embodiment of a SIGLEC-BMS protein comprises an aminoacid sequence beginning with Ala141 and ending with Ser198 as shown inFIG. 6B (SEQ ID NO:28) and is encoded by a nucleic acid molecule thathybridizes, under stringent conditions to a nucleic acid molecule thatis complementary to the nucleic acid as shown in any one of SEQ. ID NOS.1-7 and 27. Another embodiment of a SIGLEC protein comprises an aminoacid sequence that is encoded by a nucleic acid molecule thathybridizes, under stringent conditions to a nucleic acid molecule thatis complementary to the nucleic acid as shown in any one of SEQ ID NOS.1-7 and 27. Particular embodiments of the novel proteins of theinvention sequences include SIGLEC-BMS -L3a, -L3b, -L3c, -L3d, -L4,-L5a,-L5b, and -L3-995-2, (shown in SEQ ID NOS.:8-14 and 28, respectively).SIGLEC-BMS proteins may be embodied in many forms, preferably inisolated or purified form.

[0123] The SIGLEC-BMS proteins may be isolated from mammalian speciesincluding, bovine, ovine, porcine, murine, equine, and preferably human.Alternatively, purified SIGLEC-BMS proteins may be generated bysynthetic, semi-synthetic, or recombinant methods.

[0124] A skilled artisan can readily employ standard isolation andpurification methods to obtain isolated and/or purified SIGLEC proteins(Marchak, D. R., et al., 1996 in: Strategies for Protein Purificationand Characterization, Cold Spring Harbor Press, Plainview, N.Y.). Thenature and degree of isolation and purification will depend on theintended use. For example, purified SIGLEC-BMS protein molecules will besubstantially free of other proteins or molecules that impair thebinding of SIGLEC-BMS to antibodies or other ligands. Embodiments of theSIGLEC-BMS proteins include a purified SIGLEC-BMS protein or fragmentsthereof, having the biological activity of a SIGLEC-BMS protein. In oneform, such purified SIGLEC-BMS proteins, or fragments thereof, retainthe ability to bind antibody or other ligand.

[0125] In a cell, the Siglec-BMS gene sequences are predicted to includesignal peptide sequences and introns, therefore it is expected that thecell will produce various forms of a particular SIGLEC-BMS protein as aresult of post-translational modification. For example, various forms ofisolated, SIGLEC-BMS proteins may include: precursor forms that includethe signal peptide, mature forms that lack the signal peptide, anddifferent mature forms of a SIGLEC-BMS protein that result frompost-translational events such as intramolecular cleavage.

[0126] The present invention provides isolated and purified proteins,polypeptides, and fragments thereof, having an amino acid sequenceidentical to the predicted sequence of the SIGLEC-BMS sequencesdisclosed herein. Accordingly, the amino acid sequences may be identicalto a particular SIGLEC-BMS sequence, as described in any of SEQ ID NOS.:8-14, or 28.

[0127] The present invention also includes proteins having sequencevariations from the predicted SIGLEC-BMS protein sequences disclosedherein (e.g., FIGS. 2B, 3B, 4B, 5B, 7B, 8B, 9B, and 6B; SEQ ID NOS.:8-14, or 28). For example, the proteins having the variant sequencesinclude allelic variants, mutant variants, conservative substitutionvariants, and SIGLEC-BMS proteins isolated from other mammalianorganisms. The amino acid sequences may be similar to the disclosedsequences. For example, two protein sequences are considered to besimilar to each other when the percentage of amino acid residues thatdiffer between the two sequences is between about 60% to 99.99%.

[0128] The present invention encompasses mutant alleles of Siglec-BMSthat encode mutant forms of SIGLEC-BMS proteins having one or more aminoacid substitutions, insertions, deletions, truncations, or frame shifts.Such mutant forms of proteins typically do not exhibit the samebiological activity as wild-type proteins. The mutant alleles ofSiglec-BMS may or may not encode a SIGLEC-BMS protein having the samebiological activity as wild-type SIGLEC-BMS proteins, such asfunctioning as a cell adhesion molecule.

[0129] Another variant of SIGLEC-BMS proteins may have amino acidsequences that differ by one or more amino acid substitutions. Thevariant may have conservative amino acid changes, where a substitutedamino acid has similar structural or chemical properties, such asreplacement of leucine with isoleucine. Alternatively, a variant mayhave nonconservative amino acid changes, such as replacement of aglycine with a tryptophan. Similar minor variations may also includeamino acid deletions or insertions, or both. Guidance in determiningwhich and how many amino acid residues may be substituted, inserted ordeleted may be found using computer programs well known in the art, forexample, DNASTAR software.

[0130] Conservative amino acid substitutions can frequently be made in aprotein without altering either the conformation or the biologicalactivity of the protein. Such changes include substituting any ofisoleucine (I), valine (V), and leucine (L) for any other of thesehydrophobic amino acids; aspartic acid (D) for glutamic acid (E) andvice versa; glutamine (Q) for asparagine (N) and vice versa; and serine(S) for threonine (T) and vice versa. Other substitutions can also beconsidered conservative, depending on the environment of the particularamino acid and its role in the three-dimensional structure of theprotein. For example, glycine (G) and alanine (A) can frequently beinterchangeable, as can alanine (A) and valine (V). Methionine (M),which is relatively hydrophobic, can frequently be interchanged withleucine and isoleucine, and sometimes with valine. Lysine (K) andarginine (R) are frequently interchangeable in locations in which thesignificant feature of the amino acid residue is its charge and thediffering pK's of these two amino acid residues are not significant.Still other changes can be considered conservative in particularenvironments.

[0131] The proteins of the invention exhibit the biological activitiesof a SIGLEC-BMS protein, such as, for example, the ability to elicit thegeneration of antibodies that specifically bind an epitope associatedwith SIGLEC-BMS proteins. Accordingly, the SIGLEC-BMS protein, or anyoligopeptide thereof, is capable of inducing a specific immune responsein appropriate animals or cells, and/or binding with specificantibodies.

[0132] The SIGLEC-BMS Extracellular Domains

[0133] The present invention provides isolated proteins having theextracellular and/or the cytoplasmic domains of the SIGLEC-BMS proteins.

[0134] The extracellular domain of SIGLEC-BMS proteins comprisesmultiple Ig-like domains (FIG. 1). The full-length SIGLEC-BMS protein(SIGLEC-BMS-L3, also designated as SIGLEC-BMS-L3-995-2) contains fiveIg-like domains, Ig-D1 (V-set, Ser14 through Thr140), Ig-D2 (C-set,Ala141 through Ala235), Ig-D3 (C-set, Ala252 through Gln341), Ig-D4(C-set, Val358 through His443), and Ig-D5 (C-set, Tyr444 through Pro538)(FIG. 25).

[0135] The extracellular domains of known Siglec proteins (e.g., CD33)are postulated to bind with sialyated cell surface glycans (Kelm, S., etal., 1996 supra; Kelm, S., et al., 1998 supra; Vinson, M., et al., 1996supra) and mediate cell adhesion or cell signaling. To determine if theextracellular domain of SIGLEC-BMS proteins bind with sialyated cellsurface glycans, various protein binding analyses may be performed. Thebinding analyses include methods, such as fluorescence-activated cellsorting (e.g., FACs), ELISA analysis, and cell binding analysis.

[0136] The FACs analyses are conducted using full-length SIGLEC-BMSproteins, fragments thereof, a SIGLEC-BMS fusion protein, or a mutantSIGLEC-BMS protein. The preferred method includes using polypeptideshaving the extracellular domains of SIGLEC-BMS, such as the fusionproteins described in FIGS. 23 or 24. The binding studies are performedby reacting populations of mixed white blood cells, or hemopoietic celllines with polypeptides having the extracellular domains of theSIGLEC-BMS proteins.

[0137] The binding specificity of SIGLEC-BMS proteins is also determinedusing a solid support method. The SIGLEC-BMS proteins are immobilized ona solid support, such as an ELISA plate. The SIGLEC-BMS proteins usedinclude full-length SIGLEC-BMS proteins, fragments thereof, a SIGLEC-BMSfusion protein, or mutant SIGLEC-BMS protein. The cells are pre-treatedwith sialidase. The immobilized proteins are reacted with cells or celllines including: mixed white blood cells, mixed granulocytes, B cells, Tcells, NK cells, and monocytes.

[0138] Alternatively, the binding specificity of the SIGLEC-BMS proteinsis analyzed by reacting various cell types or cell lines with cells thatexpress the SIGLEC-BMS proteins of the invention. The protein-expressingcells are generated using methods well known in the art, includingmethods that result in transient or long-term expression of theSIGLEC-BMS proteins. The protein-expressing cells may be mammalian,insect, plant, bacterial, or yeast cells. The protein-expressing cellsmay express full-length SIGLEC-BMS proteins, or a fragment thereof, aSIGLEC-BMS fusion protein, or a mutant SIGLEC-BMS protein. Theprotein-expressing cells are reacted with various cell types or celllines, including: mixed white blood cells, mixed granulocytes, B cells,T cells, NK cells, and monocytes. The reacting cells are pre-treatedwith sialidase.

[0139] The SIGLEC-BMS Cytoplasmic Domains

[0140] The cytoplasmic domain of known Siglec proteins have tyrosineresidues within ITAM or ITIM motifs which mediate phosphorylation withina cell. For example, the cytoplasmic tail of Siglec-3 (e.g., CD33)includes two ITIM motifs that recruit SHP-1 and SHP-2 uponphosphorylation (Taylor, V., et al., 1999 supra).

[0141] To determine if the cytoplasmic tail domain of the SIGLEC-BMSproteins mediates phosphorylation, various methods may be performed. Themethods include kinase assays.

[0142] The kinase assays are conducted by reacting SIGLEC-BMS proteinswith kinases which provide the phosphorylation activity. The kinases arereacted with SIGLEC-BMS proteins, including full-length SIGLEC-BMSproteins, fragments thereof, a SIGLEC-BMS fusion protein, or a mutantSIGLEC-BMS protein.

[0143] The mutant SIGLEC-BMS protein may include specific substitutionof one or more amino acids within the cytoplasmic domain of a SIGLEC-BMSprotein, e.g., mutation of a specific amino acid such as a tyrosine to aphenylalanine, leucine, tryptophan, or Thr (FIG. 15). Examples of mutantSIGLEC-BMS proteins include, but are not limited to SIGLEC-BMS proteinswherein at least one tyrosine at positions 597, 641, 667, or 691 issubstituted with a phenylalanine as shown in FIG. 15, and described inExample 12.

[0144] Knowing which particular mutations in the cytoplasmic tail of aSIGLEC-BMS protein affect phosphorylation by various tyrosine kinasespermits one skilled in the art to develop methods for screening ligandsthat affect SIGLEC-mediated cell signaling. For example, SIGLEC-mediatedcell signalling can be mediated when tyrosine in any of positions 597,641, 667, or 691, of FIG. 6b, is substituted with phenylalanine,leucine, tryptophan and threonine. Additionally, ligands that bind tothe site so mutated within the cytoplasmic domain can be modified so asto modulate SIGLEC-mediated cell signalling, i.e., upregulating ordownregulating cell signalling.

[0145] Methods For Generating SIGLEC-BMS Proteins

[0146] The SIGLEC-BMS proteins of the invention may be generated byrecombinant methods. Recombinant methods are preferred if a high yieldis desired. Recombinant methods involve expressing the cloned gene in asuitable host cell. For example, a host cell is introduced with anexpression vector having a Siglec-BMS sequence, then the host cell iscultured under conditions that permit in vivo production of theSIGLEC-BMS protein encoded by the sequence.

[0147] For example, in general terms, the production of recombinantSIGLEC-BMS proteins can involve a host/vector system and the followingsteps. A nucleic acid molecule can be obtained that encodes a SIGLEC-BMSprotein or a fragment thereof, such as any one of the polynucleotidesdisclosed in SEQ ID NOs.: 1-7, or 27. The SIGLEC-BMS-encoding nucleicacid molecule can be then preferably inserted into an expression vectorin operable linkage with suitable expression control sequences, asdescribed above, to generate an expression vector containing theSIGLEC-BMS-encoding sequence. The expression vector can be introducedinto a suitable host, by standard transformation methods, and theresulting transformed host is cultured under conditions that allow theproduction and retrieval of the SIGLEC-BMS protein. For example, ifexpression of the SIGLEC-BMS gene is under the control of an induciblepromoter, then suitable growth conditions include the appropriateinducer. The SIGLEC-BMS protein, so produced, is isolated from thegrowth medium or directly from the cells; recovery and purification ofthe protein may not be necessary in some instances where some impuritiesmay be tolerated. A skilled artisan can readily adapt an appropriatehost/expression system known in the art (Cohen, et al., supra; Maniatiset al., supra) for use with SIGLEC-BMS-encoding sequences to produce aSIGLEC-BMS protein.

[0148] The SIGLEC-BMS proteins of the invention, and fragments thereof,can be generated by chemical synthesis methods. The principles of solidphase chemical synthesis of polypeptides are well known in the art andmay be found in general texts relating to this area (Dugas, H. andPenney, C. 1981 Bioorganic Chemistry, pp 54-92, Springer-Verlag, N.Y.).SIGLEC-BMS polypeptides may be synthesized by solid-phase methodologyutilizing an Applied Biosystems 430A peptide synthesizer (AppliedBiosystems, Foster City, Calif.) and synthesis cycles supplied byApplied Biosystems. Protected amino acids, such ast-butoxycarbonyl-protected amino acids, and other reagents arecommercially available from many chemical supply houses.

[0149] The present invention provides derivative protein molecules, suchas chemically modified proteins. Illustrative of such modificationswould be replacement of hydrogen by an alkyl, acyl, or amino group. TheSIGLEC-BMS protein derivatives retain the biological activities ofnatural SIGLEC-BMS proteins.

[0150] Recombinant Nucleic Acid Molecules Encoding SIGLEC-BMS

[0151] Also provided are recombinant DNA molecules (rDNAs) that includenucleotide sequences that encode SIGLEC-BMS proteins, or a fragmentthereof, as described herein. As used herein, a rDNA molecule is a DNAmolecule that has been subjected to molecular manipulation in vitro.Methods for generating rDNA molecules are well known in the art, forexample, see Sambrook et al., Molecular Cloning (1989). In the preferredrDNA molecules of the present invention, the sequences that encode theSIGLEC-BMS proteins or fragments of SIGLEC, are operably linked to oneor more expression control sequences and/or vector sequences.

[0152] Vectors

[0153] The nucleic acid molecules of the invention may be recombinantmolecules each comprising the sequence, or portions thereof, of aSiglec-BMS sequence linked to a non-Siglec-BMS sequence. For example,the Siglec-BMS sequence may be fused operatively to a vector to generatea recombinant molecule.

[0154] The term vector includes, but is not limited to, plasmids,cosmids, and phagemids. A preferred vector will be an autonomouslyreplicating vector comprising a replicon that directs the replication ofthe rDNA within the appropriate host cell. Alternatively, the preferredvector directs integration of the recombinant vector into the host cell.Various viral vectors may also be used, such as, for example, a numberof well known retroviral and adenoviral vectors (Berkner 1988Biotechniques 6:616-629).

[0155] The preferred vectors permit expression of the Siglec-BMStranscript or polypeptide sequences in prokaryotic or eukaryotic hostcells. The preferred vectors include expression vectors, comprising anexpression control element, such as a promoter sequence, which enablestranscription of the inserted Siglec-BMS sequences and can be used forregulating the expression (e.g., transcription and/or translation) of anoperably linked Siglec-BMS sequence in an appropriate host cell, such asEscherichia coli.

[0156] Expression control elements are known in the art and include, butare not limited to, inducible promoters, constitutive promoters,secretion signals, enhancers, transcription terminators, and othertranscriptional regulatory elements. Other expression control elementsthat are involved in translation are known in the art, and include theShine-Dalgarno sequence (e.g., prokaryotic host cells), and initiationand termination codons.

[0157] Specific initiation signals may also be required for efficienttranslation of a Siglec-BMS sequence. These signals include theATG-initiation codon and adjacent sequences. In cases where theSiglec-BMS initiation codon and upstream sequences are inserted into theappropriate expression vector, no additional translational controlsignals may be needed. However, in cases where only the coding sequence,or a portion thereof, is inserted, exogenous transcriptional controlsignals including the ATG-initiation codon must be provided.Furthermore, the initiation codon must be in the correct reading-frameto ensure transcription of the entire insert. Exogenous transcriptionalelements and initiation codons can be of various origins, both naturaland synthetic. The efficiency of expression may be enhanced by theinclusion of enhancers appropriate to the cell system in use (Scharf,D., et al, 1994 Results Probl. Cell. Differ. 20:125-62; Bittner, et al.,1987 Methods in Enzymol. 153:516-544).

[0158] The preferred vectors for expression of the Siglec-BMS sequencesin eukaryote host cells include expression control elements, such as thebaculovirus polyhedrin promoter for expression in insect cells. Otherexpression control elements include promoters or enhancers derived fromthe genomes of plant cells (e.g., heat shock, RUBISCO, storage proteingenes), viral promoters or leader sequences or from plant viruses, andpromoters or enhancers from the mammalian genes or from mammalianviruses.

[0159] The preferred vector includes at least one selectable marker genethat encodes a gene product that confers drug resistance such asresistance to ampicillin or tetracyline. The vector also comprisesmultiple endonuclease restriction sites that enable convenient insertionof exogenous DNA sequences. Methods for generating a recombinantexpression vector encoding the SIGLEC-BMS proteins of the invention arewell known in the art, and can be found in Maniatis, T., et al., (1989Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.) and Ausubel et al. (1989 Current Protocols inMolecular Biology, John Wiley & Sons, New York N.Y.).

[0160] The preferred vectors for generating Siglec-BMS transcriptsand/or the encoded SIGLEC-BMS polypeptides are expression vectors whichare compatible with prokaryotic host cells. Prokaryotic cell expressionvectors are well known in the art and are available from severalcommercial sources. For example, pET vectors (e.g., pET-21, NovagenCorp.), BLUESCRIPT phagemid (Stratagene, LaJolla, Calif.), pSPORT (GibcoBRL, Rockville, Md.), or ptrp-lac hybrids may be used to expressSIGLEC-BMS polypeptides in bacterial host cells.

[0161] Alternatively, the preferred expression vectors for generatingSiglec-BMS transcripts and/or the encoded SIGLEC-BMS polypeptides areexpression vectors which are compatible with eukaryotic host cells. Themore preferred vectors are those compatible with vertebrate cells.Eukaryotic cell expression vectors are well known in the art and areavailable from several commercial sources. Typically, such vectors areprovided containing convenient restriction sites for insertion of thedesired DNA segment. Typical of such vectors are PSVL and pKSV-10(Pharmacia), pBPV-1/pML2d (International Biotechnologies, Inc.), pTDT1(ATCC, #31255), and similar eukaryotic expression vectors.

[0162] Host-Vector Systems

[0163] The invention further provides a host-vector system comprising avector, plasmid, phagemid, or cosmid comprising a Siglec-BMS nucleotidesequence, or a fragment thereof, introduced into a suitable host cell. Avariety of expression vector/host systems may be utilized to carry andexpress Siglec-BMS sequences. The host-vector system can be used toexpress (e.g., produce) the SIGLEC-BMS polypeptides encoded bySiglec-BMS nucleotide sequences. The host cell can be either prokaryoticor eukaryotic. Examples of suitable prokaryotic host cells includebacteria strains from genera such as Escherichia, Bacillus, Pseudomonas,Streptococcus, and Streptomyces. Examples of suitable eukaryotic hostcells include yeast cells, plant cells, or animal cells such asmammalian cells. A preferred embodiment provides a host-vector systemcomprising the pcDNA3 vector (Invitrogen, Carlsbad, Calif.) in COS7mammalian cells, pGEX vector (Promega, Madison, Wis.) in bacterialcells, or pFastBac vector (Gibco/BRL, Rockville, Md.) in Sf9 insectcells.

[0164] Introduction of the recombinant DNA molecules of the presentinvention into an appropriate host cell is accomplished by well knownmethods that depend on the type of vector used and host system employed.For example, prokaryotic host cells are introduced (e.g., transformed)with nucleic acid molecules by electroporation or salt treatmentmethods, see for example, Cohen et al., 1972 Proc Acad Sci USA 69:2110;Maniatis, T., et al., 1989 Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y. Vertebrate cells aretransformed with vectors containing recombinant DNAs by various methods,including electroporation, cationic lipid or salt treatment (Graham etal., 1973 Virol 52:456; Wigleretal., 1979 Proc Natl Acad Sci USA76:1373-76).

[0165] Successfully transformed cells, i.e., cells that contain a rDNAmolecule of the present invention, can be identified by techniques wellknown in the art. For example, cells resulting from the introduction ofrecombinant DNA of the present invention are selected and cloned toproduce single colonies. Cells from those colonies are harvested, lysedand their DNA content examined for the presence of the rDNA using amethod such as that described by Southern, J Mol Biol (1975) 98:503, orBerent et al., Biotech (1985) 3:208, or the proteins produced from thecell assayed via a biochemical assay or immunological method.

[0166] In bacterial systems, a number of expression vectors may beselected depending upon the use intended for the SIGLEC-BMS proteins.For example, when large quantities of SIGLEC-BMS proteins are needed forthe induction of antibodies, vectors that direct high level expressionof fusion proteins that are soluble and readily purified may bedesirable. Such vectors include, but are not limited to, themultifunctional E. coli cloning and expression vectors such asBLUESCRIPT (Stratagene), in which the Siglec-BMS sequence may be ligatedinto the vector in-frame with sequences for the amino-terminal Met andthe subsequent 7 residues of β-galactosidase so that a hybrid protein isproduced; pIN vectors (Van Heeke & Schuster (1989) J Biol Chem264:5503-5509); and the like. The pGEX vectors (Promega, Madison Wis.)may also be used to express foreign proteins as fusion proteins withglutathione S-transferase (GST). In general, such fusion proteins aresoluble and can easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. Proteins made in such systems are designed to includeheparin, thrombin or factor XA protease cleavage sites so that thecloned protein of interest can be released from the GST moiety at will.

[0167] In yeast, Saccharomyces cerevisiae, a number of vectorscontaining constitutive or inducible promoters such as beta-factor,alcohol oxidase and PGH may be used. For reviews, see Ausubel et al(supra) and Grant et al (1987) Methods in Enzymology 153:516-544.

[0168] In cases where plant expression vectors are used, the expressionof a sequence encoding SIGLEC-BMS protein can be driven by any of anumber of promoters. For example, viral promoters such as the 35S and19S promoters of CaMV (Brisson, et al., (1984) Nature 310:511-514) maybe used alone or in combination with the omega leader sequence from TMV(Takamatsu, et al., (1987) EMBO J 6:307-311). Alternatively, plantpromoters such as the small subunit of RUBISCO (Coruzzi et al (1984)EMBO J 3:1671-1680; Broglie et al (1984) Science 224:838-843); or heatshock promoters (Winter J and Sinibaldi R M (1991) Results Probl CellDiffer 17:85-105) can be used. These constructs can be introduced intoplant cells by direct DNA transformation or pathogen-mediatedtransfection. For reviews of such techniques, see Hobbs, S. in: McGrawYearbook of Science and Technology (1992) McGraw Hill New York N.Y., pp191-196; or Weissbach and Weissbach (1988) in: Methods for PlantMolecular Biology, Academic Press, New York N.Y., pp 421-463.

[0169] An alternative expression system that can be used to expressSIGLEC-BMS proteins is an insect system. In one such system, Autographacaliformica nuclear polyhedrosis virus (AcNPV) can be used as a vectorto express foreign genes in Spodoptera frugiperda cells or inTrichoplusia larvae. The sequence encoding a SIGLEC-BMS protein can becloned into a nonessential region of the virus, such as the polyhedringene, and placed under control of the polyhedrin promoter. Successfulinsertion of a Siglec-BMS nucleotide sequence will render the polyhedringene inactive and produce recombinant virus lacking coat protein. Therecombinant viruses can then used to infect S. frugiperda cells orTrichoplusia larvae in which SIGLEC-BMS protein can be expressed (Smithet al (1983) J Virol 46:584; Engelhard E. K., et al, 1994 ProcNatAcadSci 91:3224-7).

[0170] In mammalian host cells, a number of viral-based expressionsystems can be utilized. In cases where an adenovirus is used as anexpression vector, a Siglec-BMS sequence can be ligated into anadenovirus transcription/translation vector consisting of the latepromoter and tripartite leader sequence. Insertion in a nonessential E1or E3 region of the viral genome results in a viable virus capable ofexpressing a SIGLEC-BMS protein in infected host cells (Logan and Shenk1984 Proc Natl Acad Sci 81:3655-59). In addition, transcriptionenhancers, such as the rous sarcoma virus (RSV) enhancer, can be used toincrease expression in mammalian host cells.

[0171] In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of theprotein include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation and acylation.Post-translational processing which cleaves a precursor form of theprotein (e.g., a prepro protein) may also be important for correctinsertion, folding and/or function. Different host cells such as CHO,HeLa, MDCK, 293, W138, etc. have specific cellular machinery andcharacteristic mechanisms for such post-translational activities and maybe chosen to ensure the correct modification and processing of theintroduced, foreign protein.

[0172] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines that stablyexpress SIGLEC-BMS proteins can be transformed using expression vectorsthat contain viral origins of replication or endogenous expressionelements and a selectable marker gene. Following the introduction of thevector, cells can be grown in an enriched media before they are switchedto selective media. The purpose of the selectable marker is to conferresistance to selection, and its presence allows growth and recovery ofcells which successfully express the introduced sequences. Resistantclumps of stably transformed cells can be proliferated using tissueculture techniques appropriate for the cell type used.

[0173] Any number of selection systems may be used to recovertransformed cell lines. These include, but are not limited to, theherpes simplex virus thymidine kinase (Wigler, M., et al., 1977 Cell11:223-32) and adenine phosphoribosyltransferase (Lowy, I. et al., 1980Cell 22:817-23) genes which can be employed in tk-minus or aprt-minuscells, respectively. Also, antimetabolite, antibiotic or herbicideresistance can be used as the basis for selection; for example, dhfrwhich confers resistance to methotrexate (Wigler, M., et al., 1980 ProcNatl Acad Sci 77:3567-70); npt, which confers resistance to theaminoglycosides neomycin and G-418 (Colbere-Garapin, F., et al., 1981 J.Mol. Biol. 150:1-14) and als or pat, which confer resistance tochlorsulfuron and phosphinotricin acetyltransferase, respectively(Murry, supra). Additional selectable genes have been described, forexample, trpB, which allows cells to utilize indole in place oftryptophan, or hisD, which allows cells to utilize histinol in place ofhistidine (Hartman, S. C. and R. C. Mulligan 1988 Proc. Natl. Acad. Sci.85:8047-51). Recently, the use of visible markers has gained popularitywith such markers as anthocyanins, β-glucuronidase and its substrate,GUS, and luciferase and its substrate, luciferin, being widely used notonly to identify transformants, but also to quantify the amount oftransient or stable protein expression attributable to a specific vectorsystem (Rhodes, C. A., et al., 1995 Methods Mol. Biol. 55:121-131).

[0174] Antibodies Reactive Against SIGLEC-BMS Proteins and Polypeptides

[0175] The invention further provides antibodies, such as polyclonal,monoclonal, chimeric, fragments, and humanized antibodies, that bind toSIGLEC-BMS proteins or fragments of SIGLEC-BMS proteins thereof.Particular examples of monoclonal antibodies of the invention are thosedesignated Siglec-10-9, Siglec-10-13, Siglec-10-14, Siglec-10-27, andSiglec-10-61, which collectively were deposited on Jul. 18, 2001 withthe American Type Culture Collection (ATCC), 10801 University Blvd.,Manassas, Va. 20110-2209 under the provisions of the Budapest Treaty,and accorded ATCC accession number (______). These antibodies can beeasily separated from the collective deposit by standard separationtechniques such as subcloning or isotype separation (Harlow, E. andLane, D. 1988 Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.).

[0176] Mabs Siglec-10-9, Siglec-10-13, Siglec-10-14, Siglec-10-27, andSiglec-10-61 all recognize and bind Siglec-10 and display different orsimilar isotypes. For example, the isotype of Siglec-10-9 is IgG3 kappaisotype, Siglec-10-13 is IgG2b kappa isotype, Siglec-10-14 is IgG1 kappaisotype, Siglec-10-27 is IgG1 kappa isotype, and Siglec-10-61 is IgG2akappa isotype.

[0177] Preferably, the antibodies of the invention bind specifically topolypeptides having SIGLEC-BMS sequences. For example, the antibodies ofthe invention can recognize and bind to a SIGLEC-BMS protein comprisingan amino acid sequence beginning with Ala141 and ending with Ser198 asshown in FIG. 6B (SEQ ID NO:28). In another embodiment, the antibody ofthe invention can recognizes and binds a SIGLEC-BMS protein comprisingan amino acid sequence beginning with Ala141 and ending with Ser198 asshown in FIG. 6B (SEQ ID NO:28) and is encoded by a nucleic acidmolecule that hybridizes, under stringent conditions to a nucleic acidmolecule that is complementary to the nucleic acid as shown in any oneof SEQ. ID NOS. 1-7 and 27. Additionally, the antibody of the inventioncan recognize and bind a SIGLEC protein comprising an amino acidsequence that is encoded by a nucleic acid molecule that hybridizes,under stringent conditions to a nucleic acid molecule that iscomplementary to the nucleic acid as shown in any one of SEQ ID NOS. 1-7and 27. Preferably, the antibody of the invention can recognize and bindSIGLEC-BMS-L3a, -L3b, -L3c, -L3d, -L4a, -L5a, -L5b, and -L3-995-2proteins (FIGS. 2B, 3B, 4B, 5B, 7B, 8B, 9B, and 6B).

[0178] Most preferably, a SIGLEC-BMS antibody specifically bind to theextracellular domain of a SIGLEC-BMS protein. The extracellular domaincan be any or all of the Ig-like domains of Siglec-10. Specifically, theantibody can recognize and bind the second Ig-like (Ig-D2) domain(Ala14l-Ser198) or the Ig-D5 domain (Tyr444-Pro538) as shown in FIG. 25.In other embodiments, the antibodies of the invention specifically bindto other domains of a SIGLEC-BMS protein or precursor, for example theantibodies bind to the cytoplasmic domain of SIGLEC-BMS proteins. Forexample, the cytoplasmic domain can encompass amino acids Lys576 throughGln697 as shown in FIG. 6B.

[0179] The most preferred antibodies will selectively bind to SIGLEC-BMSproteins and will not bind (or will bind weakly) to non-SIGLEC-BMSproteins. These antibodies can be from any source, e.g., rabbit, sheep,rat, dog, cat, pig, horse, mouse and human.

[0180] As will be understood by those skilled in the art, the regions orepitopes of a SIGLEC-BMS protein to which an antibody is directed mayvary with the intended application. For example, antibodies intended foruse in an immunoassay for the detection of membrane-bound SIGLEC-BMS onviable cells should be directed to an accessible epitope such as theextracellular domain of SIGLEC-BMS proteins. Anti-SIGLEC-BMS mAbs can beused to stain the cell surface of SIGLEC-BMS-positive cells. Thepredicted extracellular domain of SIGLEC-BMS proteins representpotential markers for screening, diagnosis, prognosis, and follow-upassays and imaging methods. In addition, SIGLEC-BMS proteins may beexcellent targets for therapeutic methods such as targeted antibodytherapy, immunotherapy, and gene therapy to treat conditions associatedwith the presence or absence of SIGLEC-BMS proteins. Antibodies thatrecognize other epitopes may be useful for the identification ofSIGLEC-BMS within damaged or dying cells, for the detection of secretedSIGLEC-BMS proteins or fragments thereof. Additionally, some of theantibodies of the invention may be internalizing antibodies, whichinternalize (e.g., enter) into the cell upon or after binding.Internalizing antibodies are useful for inhibiting cell growth and/orinducing cell death.

[0181] The invention includes any monoclonal antibody, theantigen-binding region of which competitively inhibits theimmunospecific binding of any of the monoclonal antibodies of theinvention to its target antigen. These monoclonal antibodies may beidentified by routine competition assays using, for example, any of theantibodies Siglec-10-9, Siglec-10-13, Sigle10-14, Siglec-10-27, andSiglec-10-61 (Harlow, E. and Lane, D. 1988 Antibodies, A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).Further, the invention provides recombinant proteins comprising theantigen-binding region of any the monoclonal antibodies of theinvention.

[0182] The invention also encompasses antibody fragments thatspecifically recognize a SIGLEC-BMS protein or a fragment thereof. Asused herein, an antibody fragment is defined as at least a portion ofthe variable region of the immunoglobulin molecule that binds to itstarget, i.e., the antigen binding region. Some of the constant region ofthe immunoglobulin may be included. Fragments of the monoclonalantibodies or the polyclonal antisera include Fab, F(ab′)₂, Fvfragments, single-chain antibodies, and fusion proteins which includethe immunologically significant portion (i.e., a portion that recognizesand binds SIGLEC-BMS).

[0183] The chimeric antibodies of the invention are immunoglobulinmolecules that comprise at least two antibody portions from differentspecies, for example a human and non-human portion. Chimeric antibodiesare useful, as they are less likely to be antigenic to a human subjectthan antibodies with non-human constant regions and variable regions.The antigen combining region (variable region) of a chimeric antibodycan be derived from a non-human source (e.g. murine) and the constantregion of the chimeric antibody, which confers biological effectorfunction to the immunoglobulin, can be derived from a human source(Morrison et al., 1985 Proc. Natl. Acad. Sci. U.S.A. 81:6851; Takeda etal., 1985 Nature 314:452; Cabilly et al., U.S. Pat. No. 4,816,567; Bosset al., U.S. Pat. No. 4,816,397). The chimeric antibody may have theantigen binding specificity of the non-human antibody molecule and theeffector function conferred by the human antibody molecule.

[0184] The chimeric antibodies of the present invention also compriseantibodies which are chimeric proteins, having several distinct antigenbinding specificities (e.g. anti-TNP: Boulianne et al., 1984 Nature312:643; and anti-tumor antigens: Sahagan et al., 1986 J. Immunol.137:1066). The invention also provides chimeric proteins havingdifferent effector functions (Neuberger et al., 1984 Nature 312:604),immunoglobulin constant regions from another species and constantregions of another immunoglobulin chain (Sharon et al., 1984 Nature309:364); Tan et al., 1985 J. Immunol. 135:3565-3567). Additionalprocedures for modifying antibody molecules and for producing chimericantibody molecules using homologous recombination to target genemodification have been described (Fell et al., 1989 Proc. Natl. Acad.Sci. USA 86:8507-8511).

[0185] Humanized antibodies directed against SIGLEC-BMS proteins arealso useful. As used herein, a humanized SIGLEC-BMS antibody is animmunoglobulin molecule which is capable of binding to a SIGLEC-BMSprotein. A humanized SIGLEC-BMS antibody includes variable regionshaving substantially the amino acid sequence of a human immunoglobulinand the hyper-variable region having substantially the amino acidsequence of non-human immunoglobulin. Humanized antibodies can be madeaccording to several methods known in the art (Teng et al., 1983 Proc.Natl. Acad. Sci. U.S.A. 80:7308-7312; Kozbor et al., 1983 ImmunologyToday 4:7279; Olsson et al., 1982 Meth. Enzymol. 92:3-16).

[0186] Various methods for the preparation of antibodies are well knownin the art. For example, antibodies may be prepared by immunizing asuitable mammalian host with an immunogen such as an isolated SIGLEC-BMSprotein, peptide, fragment, or an immunoconjugated form of SIGLEC-BMSprotein (Harlow 1989, in: Antibodies, Cold Spring Harbor Press, N.Y.).In addition, fusion proteins of SIGLEC-BMS may also be used asimmunogens, such as a SIGLEC-BMS fused to -GST-, -human Ig, orHis-tagged fusion proteins. Cells expressing or overexpressingSIGLEC-BMS proteins may also be used for immunizations. Similarly, anycell engineered to express SIGLEC-BMS proteins may be used. Thisstrategy may result in the production of monoclonal antibodies withenhanced capacities for recognizing endogenous SIGLEC-BMS proteins(Harlow and Lane, 1988, in: Antibodies: A Laboratory Manual. Cold SpringHarbor Press).

[0187] The amino acid sequence of SIGLEC-BMS proteins, and fragmentsthereof, may be used to select specific regions of the SIGLEC-BMSproteins for generating antibodies. For example, hydrophobicity andhydrophilicity analyses of the SIGLEC-BMS amino acid sequence may beused to identify hydrophilic regions in the SIGLEC-BMS proteinstructure. Regions of the SIGLEC-BMS protein that show immunogenicstructure, as well as other regions and domains, can readily beidentified using various other methods known in the art (Rost, B., andSander, C. 1994 Protein 19:55-72), such as Chou-Fasman, Garnier-Robson,Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf analysis.Fragments including these residues are particularly suited in generatinganti-SIGLEC-BMS antibodies.

[0188] Methods for preparing a protein for use as an immunogen and forpreparing immunogenic conjugates of a protein with a carrier such asBSA, KLH, or other carrier proteins are well known in the art.Techniques for conjugating or joining therapeutic agents to antibodiesare well known (Arnon et al., “Monoclonal Antibodies For ImmunotargetingOf Drugs In Cancer Therapy”, in: Monoclonal Antibodies And CancerTherapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985);Hellstrom et al., “Antibodies For Drug Delivery”, in: Controlled DrugDelivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker,Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In CancerTherapy: A Review”, in: Monoclonal Antibodies '84: Biological AndClinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); andThorpe et al., “The Preparation And Cytotoxic Properties OfAntibody-Toxin Conjugates”, Immune. Rev., 62:119-58 (1982); Sodee etal., 1997, Clin. Nuc. Med. 21: 759-766). In some circumstances, directconjugation using, for example, carbodiimide reagents may be used; inother instances linking reagents such as those supplied by PierceChemical Co., Rockford, Ill., may be effective.

[0189] Administration of a SIGLEC-BMS immunogen is conducted generallyby injection over a suitable time period and with use of a suitableadjuvant, as is generally understood in the art. During the immunizationschedule, titers of antibodies can be taken to determine adequacy ofantibody formation.

[0190] While the polyclonal antisera produced in this way may besatisfactory for some applications, for pharmaceutical compositions,monoclonal antibody preparations are preferred. Immortalized cell lineswhich secrete a desired monoclonal antibody may be prepared using thestandard method of Kohler and Milstein (Nature 256: 495-497) ormodifications which effect immortalization of lymphocytes or spleencells, as is generally known. The immortalized cell lines secreting thedesired antibodies are screened by immunoassay in which the antigen isthe SIGLEC-BMS protein or a fragment thereof. When the appropriateimmortalized cell culture secreting the desired antibody is identified,the cells can be cultured either in vitro or by production in ascitesfluid. The desired monoclonal antibodies are then recovered from theculture supernatant or from the ascites supernatant.

[0191] Novel antibodies of human origin can be also made to the antigenhaving the appropriate biological functions. The completely humanantibodies are particularly desirable for therapeutic treatment of humanpatients. The human monoclonal antibodies may be made by using theantigen, e.g. a SIGLEC-BMS protein or peptide thereof, to sensitizehuman lymphocytes to the antigen in vitro, followed byEBV-transformation or hybridization of the antigen-sensitizedlymphocytes with mouse or human lymphocytes, as described by Borrebaecket al. (Proc. Natl. Acad. Sci. USA 85:3995-99 (1988)).

[0192] Alternatively, human antibodies can be produced using transgenicanimals such as mice which are incapable of expressing endogenousimmunoglobulin heavy and light chain genes, but which can express humanheavy and light chain genes. The transgenic mice are immunized in thenormal fashion with a selected antigen, e.g., all or a portion of apolypeptide of invention. Monoclonal antibodies directed against theantigen can be produced using conventional hybridoma technology. Thehuman immunoglobulin transgenes harbored by the transgenic micerearrange during B cell differentiation, and subsequently undergo classswitching and somatic mutations. Thus, using this technology, it ispossible to produce therapeutically useful IgG, IgA, and IgB antibodies.For an overview of this technology to produce human antibodies, seeLonberg and Haszar (1995, Int. Rev. Immunol. 13;65-93). A detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies can be found in U.S. Pat. Nos. 5,625,126;5,633,425; 5,569,825; 5,661,016; and 5,545,806.

[0193] The antibodies or fragments may also be produced by recombinantmeans. The antibody regions that bind specifically to the desiredregions of the SIGLEC-BMS protein can also be produced in the context ofchimeric or CDR grafted antibodies of multiple species origin.

[0194] Uses of the Molecules of the Invention

[0195] The nucleic acid molecules encoding SIGLEC-BMS proteins areuseful for a variety of purposes, including their use in diagnosisand/or prognosis methods. The nucleic acid molecules and proteins of theinvention may be used to test the presence and/or amount of Siglec-BMSnucleotide sequences and/or SIGLEC-BMS protein in a suitable biologicalsample.

[0196] The suitable biological sample can be from an animal or a human.The sample can be a cell sample or a tissue sample, including samplesfrom spleen, lymph node, thymus, bone marrow, liver, heart, brain,placenta, lung, skeletal muscle, kidney and pancreas. The sample can bea biological fluid, including, urine, blood sera, blood plasma, phlegm,or lavage fluid. Alternatively, the sample can be a swab from the nose,ear or throat.

[0197] Additionally, the SIGLEC-BMS proteins are able to elicit thegeneration of antibodies, which can serve as molecules for use invarious diagnostic or therapeutic modalities. SIGLEC-BMS proteins mayalso be used to identify and isolate agents that bind to SIGLEC-BMSproteins (e.g., SIGLEC-BMS ligands) and modulate the biological activityof SIGLEC-BMS proteins.

[0198] Uses of Nucleic Acid Molecules Encoding Siglec-BMS Proteins

[0199] The nucleic acid molecules encoding SIGLEC-BMS proteins can beused in various hybridization methods to identify and/or isolatenucleotide sequences related to the Siglec-BMS nucleotide sequencedescribed herein. Sequences related to Siglec-BMS sequence are usefulfor developing additional ligands and antibodies. The hybridizationmethods are used to identify/isolate DNA and RNA sequences that areidentical or similar to the Siglec-BMS sequences, such as SIGLEC-BMShomologues, alternatively sliced isoforms, allelic variants, and mutantforms of the SIGLEC protein, as well as their coding and gene sequences.

[0200] Full-length or fragments of the nucleotide sequences that encodethe SIGLEC-BMS proteins, described herein, can be used as a nucleic acidprobes to retrieve nucleic acid molecules having sequences related toSiglec-BMS sequences.

[0201] In one embodiment, a Siglec-BMS nucleic acid probe is used toscreen genomic libraries, such as libraries constructed in lambda phageor BACs (bacterial artificial chromosomes) or YACs (yeast artificialchromosomes), to isolate a genomic clone of a Siglec gene. Siglec-BMSsequences from genomic libraries are useful for isolating upstream ordownstream non-coding sequences, such as promoter, enhancer, andtranscription termination sequences. The upstream sequences may bejoined to non-Siglec-BMS sequences in order to construct a recombinantDNA molecule that expresses the non-Siglec-BMS sequence uponintroduction into an appropriate host cell. In another embodiment, aSiglec-BMS probe is used to screen cDNA libraries to isolate cDNA clonesexpressed in certain tissues or cell types. Siglec-BMS sequences fromcDNA libraries are useful for isolating sequences from various celltypes, tissue types, or from various mammalian subjects.

[0202] Additionally, pairs of oligonucleotide primers can be preparedfor use in a polymerase chain reaction (PCR) to selectively amplify orclone nucleic acid molecules encoding SIGLEC-BMS proteins, or fragmentsthereof. PCR methods (U.S. Pat. No. 4,965,188) that include numerouscycles of denature/anneal/polymerize steps are well known in the art andcan readily be adapted for use in isolating other SIGLEC-BMS-encodingnucleic acid molecules.

[0203] In addition, the nucleic acid molecules of the invention may alsobe employed in diagnostic embodiments, using the Siglec-BMS nucleic acidprobes to determine the presence and/or the amount of Siglec-BMSsequences present in a biological sample.

[0204] One diagnostic embodiment encompasses determining the amount ofSiglec-BMS nucleotide sequences present within asuitable biologicalsample, using a Siglec-BMS probe in a hybridization procedure.

[0205] Another embodiment encompasses quantifying the amount ofSiglec-BMS nucleic acid molecules in the biological sample from a testsubject, using a Siglec-BMS probe in a hybridization procedure. Theamount of Siglec-BMS nucleic acid molecules in the test sample can becompared with the amount of Siglec-BMS nucleic acid molecules in areference sample from a normal subject. The presence of a measurablydifferent amount of Siglec-BMS nucleic acid molecules between the testand reference samples may correlate with the presence or with theseverity of a disease associated with abnormal levels or a deficiency ofSiglec-BMS nucleic acid molecules.

[0206] In another embodiment, monitoring the amount of Siglec-BMS RNAtranscripts over time is effected by quantitatively determining theamount of Siglec-BMS RNA transcripts in test samples taken at differentpoints in time. A difference in the amounts of Siglec-BMS RNAtranscripts in the various samples being indicative of the course of adisease associated with expression of a Siglec-BMS transcripts.

[0207] As a further embodiment, the diseases or disorders associatedwith Siglec-BMS transcripts or proteins are detected by an increase ordeficiency in Siglec-BMS gene copy number. Methods for detecting genecopy number include chromosome mapping by Fluorescence In SituHybridization (FISH analysis) (Rowley et al., 1990, PNAS USA 87:9358-9362, H. Shizuya, PNAS USA, 89:8794). Methods for determining anincrease in Siglec-BMS gene copy number are important because theincrease may correlate with an increase in the severity of the diseaseassociated with SIGLEC-BMS protein and poor patient outcome.

[0208] To conduct such diagnostic methods, a suitable biological samplefrom a test subject is contacted with a Siglec-BMS probe, underconditions effective to allow hybridization between the sample nucleicacid molecules and the probe. In a similar manner, a biological samplefrom a normal subject is contacted with a Siglec-BMS probe andhybridized under similar conditions. The presence of the nucleic acidmolecules hybridized to the probe is detected. The relative and/orquantified amount of the hybridized molecules may be compared betweenthe test and reference samples. The Siglec-BMS probes are preferablylabeled with any of the known detectable labels, including radioactive,enzymatic, fluorescent, or even chemiluminescent labels.

[0209] Many suitable variations of hybridization technology areavailable for use in the detection of nucleic acids having Siglec-BMSsequences. These include, for example, Southern and Northern procedures.Other hybridization techniques and systems are known that can be used inconnection with the detection aspects of the invention, includingdiagnostic assays such as those described in Falkow et al., U.S. Pat.No. 4,358,535. Another hybridization procedure includes in situhybridization, where the target nucleic acids are located within one ormore cells and are contacted with the Siglec-BMS probes. As is wellknown in the art, the cells are prepared for hybridization by fixation,e.g. chemical fixation, and placed in conditions that permithybridization of the Siglec-BMS probe with nucleic acids located withinthe fixed cell.

[0210] Alternatively, Siglec-BMS nucleic acids are separated from a testsample prior to contact with a probe. The methods for isolating targetnucleic acids from the sample are well known, and include cesiumchloride gradient centrifugation, chromatography (e.g., ion, affinity,magnetic), and phenol extraction.

[0211] Uses of SIGLEC-BMS Proteins

[0212] SIGLEC-BMS proteins are expressed in eosinophils, neutrophils andmonocytes and the expression of these molecules is immune-restricted,indicating that these proteins may be involved in modulating eosinophilor other immune cell maturation, migration, activation, or communicationwith other cells. Thus, SIGLEC-BMS proteins are postulated to beinvolved in the pathogenesis of asthma and other allergic diseases,leukemia, or inflammation.

[0213] SIGLEC-BMS proteins are thus attractive targets for drugdevelopment. Drugs directed against SIGLEC-BMS will likely inhibitinflammation, tissue damage and remodeling in asthma and possibly otherinflammatory diseases such as allergic rhinitis, osteoarthritis,inflammatory bowel disease, Crohn's disease, chronic obstructivepulmonary disease, psoriasis, conjunctivitis, glomerular nephritis,rheumatoid arthritis and gingivitis. In addition, given that previouslydiscovered SIGLEC proteins have been detected on circulating, immaturewhite blood cells in some types of monomyelocytic leukemias (Elghetany,M. T. 1998 Haematologica 83:1104-1115), it is likely that drugs directedagainst SIGLEC-BMS proteins could be used to treat or target certaintypes of leukemia (e.g., eosinophilic leukemia).

[0214] In addition, the SIGLEC-BMS proteins and fragments of theinvention can be used to elicit the generation of antibodies thatspecifically bind an epitope associated with SIGLEC-BMS protein, asdescribed herein (Kohler and Milstein, supra). The SIGLEC-BMS antibodiesinclude fragments, such Fv, Fab′, and F(ab′)2. SIGLEC-BMS antibodieswhich are immunoreactive with selected domains or regions of theSIGLEC-BMS protein are particularly useful. The domains of interestinclude the extracellular and cytoplasmic domains of SIGLEC-BMSproteins.

[0215] In one embodiment, the SIGLEC-BMS antibodies are used to screenexpression libraries in order to obtain proteins related to SIGLEC-BMSproteins (e.g., homologues).

[0216] In another embodiment, SIGLEC-BMS antibodies are used to enrichor purify SIGLEC-BMS proteins from a sample having a heterologouspopulation of proteins. The enrichment and purifying methods includeconventional techniques, such as immuno-affinity methods. In general,the immuno-affinity methods include the following steps: preparing anaffinity matrix by linking a solid support matrix with SIGLEC-BMSantibodies, which linked affinity matrix specifically binds withSIGLEC-BMS proteins; contacting the linked affinity matrix with thesample under conditions that permit the SIGLEC-BMS proteins in thesample to bind to the linked affinity matrix; removing thenon-SIGLEC-BMS proteins that did not bind to the linked affinity matrix,thereby enriching or purifying for the SIGLEC-BMS proteins. A furtherstep may include eluting the SIGELC-BMS proteins from the affinitymatrix. The general steps and conditions for affinity enrichment for adesired protein or protein complex can be found in Antibodies: ALaboratory Manual (Harlow, E. and Lane, D., 1988 CSHL, Cold Spring,N.Y.).

[0217] SIGLEC-BMS antibodies are also used to detect, sort, or isolatecells expressing a SIGLEC-BMS protein. The SIGLEC-BMS-positive (+) cellsare detected within various biological samples. The presence ofSIGLEC-BMS proteins on cells (alone or in combination with other cellsurface markers) may be used to distinguish and isolate cells (e.g.,sorting) expressing SIGLEC-BMS from other cells, using antibody-basedcell sorting or affinity purification techniques. The SIGLEC-BMSantibodies may be used to generate large quantities of relatively pureSIGLEC-BMS-positive cells from individual subjects or patients, whichcan be grown in tissue culture. In this way, for example, an individualsubject's cells may be expanded from a limited biopsy sample and thentested for the presence of diagnostic and prognostic genes, proteins,chromosomal aberrations, gene expression profiles, or other relevantgenotypic and phenotypic characteristics, without the potentiallyconfounding variable of contaminating cells. Similarly, patient-specificvaccines and cellular immunotherapeutics may be created from such cellpreparations. The methods for detecting, sorting, and isolatingSIGLEC-BMS-positive cells use various imaging methodologies, such asfluorescence or immunoscintigraphy with Induim-111 (or other isotope).

[0218] There are multiple diagnostic uses of the antibodies of theinvention. For example, CD33 is upregulated in myelodysplastic syndromes(Elghetamy, 1998 supra) and is used as a diagnostic marker for leukemia.The invention provides methods for diagnosing in a subject, e.g., ananimal or human subject, a disease associated with the presence ordeficiency of the SIGLEC-BMS protein(s). In one embodiment, the methodcomprises quantitatively determining the amount of SIGLEC-BMS protein inthe sample (e.g., cell or biological fluid sample) using any one orcombination of the antibodies of the invention. Then the amount sodetermined can be compared with the amount in a sample from a normalsubject. The presence of a measurably different amount in the sample(i.e., the amount of SIGLEC-BMS proteins in the test sample exceeds oris reduced from the amount of SIGLEC-BMS proteins in a normal sample)indicates the presence of the disease.

[0219] The anti-SIGLEC-BMS antibodies of the invention may beparticularly useful in diagnostic imaging methodologies, where theantibodies have a detectable label. In accordance with the practice ofthe invention, the methods could use any monoclonal antibody thatrecognizes a SIGLEC BMS protein or fragment thereof including thoseantibodies, the antigen-binding region of which, competitively inhibitsthe immunospecific binding of any of the monoclonal antibodies Siglec10-9, Siglec 10-13, Siglec 10-14, Siglec 10-27, or Siglec 10-61, to itstarget antigen.

[0220] The invention provides various immunological assays useful forthe detection of SIGLEC-BMS proteins in a suitable biological sample.Suitable detectable markers include, but are not limited to, aradioisotope, a fluorescent compound, a bioluminescent compound,chemiluminescent compound, a chromophore, a metal chelator, biotin, oran enzyme. Such assays generally comprise one or more labeled SIGLEC-BMSantibodies that recognize and bind a SIGLEC-BMS protein, and includevarious immunological assay formats well known in the art, including butnot limited to various types of precipitation, agglutination, complementfixation, radioimmunoassays (RIA), enzyme-linked immunosorbent assays(ELISA), enzyme-linked immunofluorescent assays (ELIFA) (H. Liu et al.1998 Cancer Research 58: 4055-4060), immunohistochemical analyses andthe like.

[0221] In addition, immunological imaging methods that detect cellsexpressing SIGLEC-BMS are also provided by the invention, including butnot limited to radioscintigraphic imaging methods using labeledSIGLEC-BMS antibodies. Such assays may be clinically useful in thedetection and monitoring the number and/or location of cells expressingSIGLEC-BMS proteins.

[0222] The invention additionally provides methods of determining adifference in the amount and distribution of SIGLEC-BMS protein in atest biological sample from an afflicted subject relative to the amountand distribution in a reference sample from a normal subject. In oneembodiment, the method comprises contacting the test and referencesample with an anti-SIGLEC-BMS antibody that specifically forms acomplex with a SIGLEC-BMS protein, thereby providing a means fordetecting the difference in the amount and distribution of SIGLEC-BMS inthe test and reference samples.

[0223] Additionally, the invention provides methods for monitoring thecourse of disease or disorders associated with SIGLEC-BMS in a testsubject by measuring the amount of SIGLEC-BMS protein in a sample fromthe test subject at various points in time. This is done for purposes ofdetermining a change in the amount of SIGLEC-BMS in the sample overtime. Monitoring the course of disease or disorders may optimize thetiming, dosage, and type of treatment, over time. In one embodiment, themethod comprises quantitatively determining in a first sample from thesubject the presence of a SIGLEC-BMS protein and comparing the amount sodetermined with the amount present in a second sample from the samesubject taken at a different point in time, a difference in the amountsdetermined being indicative of the course of the disease.

[0224] One embodiment of the invention is a method for diagnosing anasthmatic condition in a candidate subject. This method comprises:obtaining a biological sample from an candidate asthmatic subject (e.g.,test sample) and from normal subjects (e.g., reference samples);contacting the test and reference sample(s) with an anti-SIGLEC-BMSantibody that specifically forms a complex with a SIGLEC-BMS protein;detecting the complex so formed in the test and reference samples;comparing the amount of complex formed in the test and referencesamples, where a measurable difference in the amount of the complexformed in the test and reference samples is indicative of an asthmaticcondition. Elevated levels of SIGLEC-BMS in the bloodstream or lavagefluid may be a way of detecting the condition or severity of asthma.This detection can be done by ELISA or similar methods using antibodiesthat react with SIGLEC-BMS proteins.

[0225] SIGLEC-BMS antibodies may also be used therapeutically tomodulate (e.g., inhibit or activate) the biological activity ofSIGLEC-BMS proteins, or to target therapeutic agents, such asanti-inflammatory drugs, to cells expressing SIGLEC-BMS proteins. Forexample, cells expressing SIGLEC-BMS can be targeted, using antibodiesthat bind with cells expressing SIGLEC-BMS proteins. The binding of theSIGLEC-BMS antibody with the cells decrease the biological activity ofSIGLEC-BMS proteins, thereby inhibiting the growth of theSIGLEC-BMS-expressing cell and decreasing the disease associated withabnormal cellular expression of SIGLEC-BMS proteins.

[0226] The SIGLEC-BMS antibodies or fragments thereof may be conjugatedto a second molecule, such as a therapeutic agent (e.g., a cytotoxicagent) resulting in an immunoconjugate. The immunoconjugate can be usedfor targeting the second molecule to a SIGLEC-BMS positive cell, therebyinhibiting the growth of the SIGLEC-BMS positive cell (Vitetta, E. S. etal., 1993 “Immunotoxin Therapy” pp. 2624-2636, in: Cancer: Principlesand Practice of Oncology, 4th ed., ed.: DeVita, Jr., V. T. et al., J.B.Lippincott Co., Philadelphia).

[0227] The therapeutic agents include, but are not limited to,anti-tumor drugs, cytotoxins, radioactive agents, cytokines, and asecond antibody or an enzyme. Examples of cytotoxic agents include, butare not limited to ricin, doxorubicin, daunorubicin, taxol, ethiduimbromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine,colchicine, dihydroxy anthracin dione, actinomycin D, diphteria toxin,Pseudomonas exotoxin (PE) A, PE40, abrin, and glucocorticoid and otherchemotherapeutic agents, as well as radioisotopes.

[0228] Further, the invention provides an embodiment wherein theantibody of the invention is linked to an enzyme that converts a prodruginto a cytotoxic drug. Alternatively, the antibody is linked to enzymes,lymphokines, or oncostatin.

[0229] Use of immunologically reactive fragments, such as Fab, Fab′, orF(ab′)₂ fragments is often preferable, especially in a therapeuticcontext, as these fragments are generally less immunogenic than thewhole immunoglobulin. The invention also provides pharmaceuticalcompositions having the monoclonal antibodies or anti-idiotypicmonoclonal antibodies of the invention, in a pharmaceutically acceptablecarrier.

[0230] Screening For SIGLEC-BMS Ligands

[0231] Another aspect of the invention relates to screening methods foridentifying agents of interest that bind with (e.g., ligands) and/ormodulate the biological activity of SIGLEC-BMS proteins. BecauseSIGLEC-BMS proteins are expressed in eosinophils, these agents may beinvolved in modulating eosinophil or other immune cell maturation,migration, activation, or communication with other cells. Thus, agentsthat bind with and modulate the biological activity of SIGLEC-BMSproteins may be effective in reducing certain symptoms of asthma andother allergic diseases, leukemia, or reduce inflammation.

[0232] Typically, the goal of such screening methods is to identify anagent(s) that binds to the target polypeptide (e.g., SIGLEC-BMS) andcauses a change in the biological activity of the target polypeptide,such as activation or inhibition of the target polypeptide, therebydecreasing diseases associated with abnormal cellular expression ofSIGLEC-BMS proteins. The agents of interest are identified from apopulation of candidate agents.

[0233] The screening methods include assays for detecting andidentifying agents, and cellular constituents that bind to SIGLEC-BMSproteins (e.g., ligands of SIGLEC-BMS). In one embodiment, a screeningassay comprises the following: contacting a SIGLEC-BMS protein with atest agent or cellular extract, under conditions that allow association(e.g., binding) of the SIGLEC-BMS protein with the test agent or acomponent of the cellular extract; and determining if a complexcomprising the agent or component associated with the SIGLEC-BMS proteinis formed. The screening methods are suitable for use in highthrough-put screening methods

[0234] The binding of an agent with a SIGLEC-BMS protein can be assayedusing a shift in the molecular weight or a change in biological activityof the unbound SIGLEC-BMS protein, or the expression of a reporter genein a two-hybrid system (Fields, S. and Song, O., 1989, Nature340:245-246). The method used to identify whether an agent/cellularcomponent binds to a SIGLEC-BMS protein is based primarily on the natureof the SIGLEC-BMS protein used. For example, a gel retardation assay isused to determine whether an agent binds to SIGLEC-BMS or a fragmentthereof. Alternatively, immunodetection and biochip (e.g., U.S. Pat. No.4,777,019) technologies are adopted for use with the SIGLEC-BMS protein.An alternative method for identifying agents that bind with SIGLEC-BMSproteins employs TLC overlay assays using glycolipid extracts fromimmune-type cells (K. M. Abdullah, et al., 1992 Infect. Immunol.60:56-62). A skilled artisan can readily employ numerous art-knowntechniques for determining whether a particular agent binds to aSIGLEC-BMS protein.

[0235] Alternatively or consecutively, the biological activity of theSIGLEC-BMS protein, as part of the complex, can be analyzed as a meansfor identifying agonists and antagonists of SIGLEC-BMS activity. Forexample, a method used to isolate cellular components that bind CD22 (D.Sgroi, et al., 1993 J. Biol. Chem. 268:7011-7018; L. D. Powell, et al.,1993 J. Biol. Chem. 268:7019-7027) is adapted to isolate cell-surfaceglycoproteins that bind to SIGLEC-BMS proteins by contacting cellextracts with an affinity column having immobilized anti-SIGLEC-BMSantibodies.

[0236] As used herein, an agent is said to antagonize SIGLEC-BMSactivity when the agent reduces the biological activity of a SIGLEC-BMSprotein. The preferred antagonist selectively antagonizes the biologicalactivity of SIGLEC-BMS, not affecting any other cellular proteins.Further, the preferred antagonist reduces SIGLEC-BMS activity by morethan 50%, more preferably by more than 90%, most preferably eliminatingall SIGLEC-BMS activity.

[0237] As used herein, an agent is said to agonize SIGLEC-BMS activitywhen the agent increases the biological activity of a SIGLEC-BMSprotein. The preferred agonist selectively agonizes the biologicalactivity of SIGLEC-BMS, not affecting any other cellular proteins.Further, the preferred antagonist increases SIGLEC-BMS activity by morethan 50%, more preferably by more than 90%, most preferably more thandoubling SIGLEC-BMS activity.

[0238] Another embodiment of the assays of the invention includesscreening agents and cellular constituents that bind to SIGLEC-BMSproteins using a yeast two-hybrid system (Fields, S. and Song, O.,supra) or using a binding-capture assay (Harlow, supra). Generally, theyeast two-hybrid system is performed in a yeast host cell carrying areporter gene, and is based on the modular nature of the GALtranscription factor which has a DNA binding domain and atranscriptional activation domain. The two-hybrid system relies on thephysical interaction between a recombinant protein that comprises theDNA binding domain and another recombinant protein that comprises thetranscriptional activation domain to reconstitute the transcriptionalactivity of the modular transcription factor, thereby causing expressionof the reporter gene. Either of the recombinant proteins used in thetwo-hybrid system can be constructed to include the SIGLEC-BMS-encodingsequence to screen for binding partners of SIGLEC-BMS. The yeasttwo-hybrid system can be used to screen cDNA expression libraries (G. J.Hannon, et al. 1993 Genes and Dev. 7: 2378-2391), and random aptmerlibraries (J. P. Manfredi, et al. 1996 Molec. And Cell. Biol. 16:4700-4709) or semi-random (M. Yang, et al. 1995 Nucleic Acids Res. 23:1152-1156) aptmers libraries for SIGLEC-BMS ligands.

[0239] SIGLEC-BMS proteins which are used in the screening assaysdescribed herein include, but are not limited to, an isolated SIGLEC-BMSprotein, a fragment of a SIGLEC-BMS protein, a cell that has beenaltered to express a SIGLEC-BMS protein, or a fraction of a cell thathas been altered to express a SIGLEC-BMS protein.

[0240] The candidate agents to be tested for binding with SIGLEC-BMSproteins and/or modulating the activity of SIGLEC-BMS proteins can be,as examples, peptides, antibody, small molecules, and vitaminderivatives, as well as carbohydrates. A skilled artisan can readilyrecognize that there is no limit as to the structural nature of theagents tested for binding to SIGLEC-BMS proteins. One class of agents ispeptide agents whose amino acid sequences are chosen based on the aminoacid sequence of the SIGLEC-BMS protein. Small peptide agents can serveas competitive inhibitors of SIGLEC-BMS protein.

[0241] Candidate agents that are tested for binding with SIGLEC-BMSproteins and/or modulating the activity of SIGLEC-BMS proteins arerandomly selected or rationally selected. As used herein, an agent issaid to be randomly selected when the agent is chosen randomly withoutconsidering the specific sequences of the SIGLEC-BMS protein. Examplesof randomly selected agents are members of a chemical library, a peptidecombinatorial library, constituents of a growth broth of an organism, orplant extract.

[0242] As used herein, an agent is said to be rationally selected whenthe agent is chosen on a nonrandom basis that is based on the sequenceof the target site (SIGLEC-BMS protein) and/or its conformation inconnection with the agent's action. Agents are rationally selected byutilizing the peptide sequences that make up the SIGLEC-BMS protein. Forexample, a rationally selected peptide agent can be a peptide whoseamino acid sequence is identical to a selected fragment of a SIGLEC-BMSprotein.

[0243] The cellular extracts to be tested for binding with SIGLEC-BMSproteins and/or modulating the activity of SIGLEC-BMS proteins are, asexamples, aqueous extracts of cells or tissues, organic extracts ofcells or tissues or partially purified cellular fractions. A skilledartisan can readily recognize that there is no limit as to the source ofthe cellular extracts used in the screening methods of the presentinvention.

[0244] Pharmaceutical Compositions of the Invention

[0245] The invention includes pharmaceutical compositions for use in thetreatment of immune system diseases comprising pharmaceuticallyeffective amounts of soluble SIGLEC-BMS molecules. The pharmaceuticalcomposition can include soluble SIGLEC-BMS protein molecules and/ornucleic acid molecules, and/or vectors encoding the molecules. In apreferred embodiment, the soluble SIGLEC-BMS protein molecule has theamino acid sequence of the extracellular domain of SIGLEC-10 as shown ineither FIG. 6B. The compositions may additionally include othertherapeutic agents, including, but not limited to, drug toxins, enzymes,antibodies, or conjugates.

[0246] In one embodiment, the pharmaceutical compositions may comprise aSIGLEC antibody, either unmodified, conjugated to a therapeutic agent(e.g., drug, toxin, enzyme or second antibody) or in a recombinant form(e.g., chimeric or bispecific). The compositions may additionallyinclude other antibodies or conjugates (e.g., an antibody cocktail).

[0247] The pharmaceutical compositions also preferably include suitablecarriers and adjuvants which include any material which when combinedwith the SIGLEC-BMS molecules of the invention retains the molecule'sactivity and is non-reactive with the subject's immune system. Examplesof suitable carriers and adjuvants include, but are not limited to,human serum albumin; ion exchangers; alumina; lecithin; buffersubstances, such as phosphates; glycine; sorbic acid; potassium sorbate;and salts or electrolytes, such as protamine sulfate. Other examplesinclude any of the standard pharmaceutical carriers such as a phosphatebuffered saline solution; water; emulsions, such as oil/water emulsion;and various types of wetting agents. Other carriers may also includesterile solutions; tablets, including coated tablets and capsules.Typically such carriers contain excipients such as starch, milk, sugar,certain types of clay, gelatin, stearic acid or salts thereof, magnesiumor calcium stearate, talc, vegetable fats or oils, gums, glycols, orother known excipients. Such carriers may also include flavor and coloradditives or other ingredients. Compositions comprising such carriersare formulated by well known conventional methods. Such compositions mayalso be formulated within various lipid compositions, such as, forexample, liposomes as well as in various polymeric compositions, such aspolymer microspheres.

[0248] The pharmaceutical compositions of the invention can beadministered to a subject using conventional modes of administrationincluding, but not limited to, intravenous (i.v.) administration,intraperitoneal (i.p.) administration, intramuscular (i.m.)administration, subcutaneous administration, oral administration,administration as a suppository, or as a topical contact, or theimplantation of a slow-release device such as a miniosmotic pump. Thepharmaceutical compositions of the invention may be in a variety ofdosage forms, which include, but are not limited to, liquid solutions orsuspensions, tablets, pills, powders, suppositories, polymericmicrocapsules or microvesicles, liposomes, and injectable or infusiblesolutions. The preferred form depends upon the mode of administrationand the therapeutic application.

[0249] The most effective mode of administration and dosage regimen forthe compositions of this invention depends upon many factors including,but not limited to the type of tissue affected, the type of autoimmunedisease being treated, the severity of the disease, a subject's health,and a subject's response to the treatment with the agents. Accordingly,dosages of the agents can vary depending on the subject and the mode ofadministration.

[0250] The soluble SIGLEC-BMS molecules may be administered to a subjectin an appropriate amount and for a suitable time period (e.g. length oftime and/or multiple times). Administration of the pharmaceuticalcompositions of the invention can be performed over various times. Inone embodiment, the pharmaceutical compositions of the invention can beadministered for one or more hours. In addition, the administration canbe repeated depending on the severity of the disease as well as otherfactors as understood in the art.

[0251] The following examples are presented to illustrate the presentinvention and to assist one of ordinary skill in making and using thesame. The methodology and results may vary depending on the intendedgoal of treatment and the procedures employed. The examples are notintended in any way to otherwise limit the scope of the invention.

EXAMPLE 1

[0252] The following provides a description of the methods used toobtain the Siglec-BMS cDNA clones, the sequences of the cDNA clones andthe SIGLEC-BMS polypeptides.

[0253] The nucleic acid molecules having Siglec-BMS nucleotide sequenceswere obtained by searching a proprietary ESTdatabase (Incyte ESTdatabase, Palo Alto, Calif.) for human gene sequences that exhibitelevated transcript expression in diseased immune tissues compared tonormal tissues, identifying the cDNA clones of interest, acquiring theclones from the proprietor of the database (Incyte), and sequencing theentire insert of the clones. In particular, the search identified anucleotide sequence, Siglec-BMS (-L3a) that is preferentially expressedin eosinophils from an asthmatic patient. Other cDNA clones having thenucleotide sequences of Siglec-BMS (-L3b, -L3c, -L3d, -L4a, -L5a, and-L5b) were obtained by further mining of the same ESTdatabase andacquiring the cDNA clones.

[0254] DNA from individual cDNA clones was isolated using a QiagenBioRobot 9600. The purified DNA was then cycle sequenced using dyeterminator chemistries and subsequently separated and detected byelectrophoresis through acrylamide gels run on ABI 377 sequencers(Perkin-Elmer).

[0255] The nucleotide sequences of the Siglec-BMS cDNA clones wereanalyzed in all 3 open reading frames (ORFs) on both strands todetermine the predicted amino acid sequence of the encoded protein. Thenucleotide sequence analysis was performed using SeqWeb version 1.1(GCG, Genetics Computer Group Wisconsin Package Version 10, Madison,Wis., 1999) using the Translate Tool to predict the amino acidsequences, and using the Structure Analysis Tool for predicting themotifs. Several Ig-like domains were identified in all clones whichallowed for further similarity analysis using the Pileup Tool in GCG(Unix version 9.1, 1997). One additional Ig domain was identified in theL3 clones, based on this similarity analysis. A comparison of the aminoacid sequences of each clone suggested that these cDNA clones includedsequences that encoded proteins having sequence homology with human CD33(Siglec-3). These nucleotide sequences were designated Siglec-BMS (SEQID NOS:1-7, 15) and the proteins sequences (SEQ ID NOS:8-14, 16) weredesignated SIGLEC-BMS.

[0256] A web-based lab management data system, PHRED, was used to trackand process the sequence data (Ewing, B., Hillier, L., Wendl, M., andGreen, P. 1998 Genome Research 8:175-185 “Basecalling of automatedsequencer traces using PHRED. I. Accuracy assessment”), and the PHRAPalgorithm was used for assembly of separate sequences into contiguouspieces (Ewing, B., and Green, P. 1998 Genome Research 8:186-194“Basecalling of automated sequencer traces using PHRED. II. Errorprobabilities”). The assembled DNA data was edited using CONSED (Gordon,D., Abajian, C. and Green, P. 1998 Genome Research 8:195-202 “Consed: Agraphical tool for sequence finishing”) to manually inspect quality andto design primers for closing sequence gaps and achieving contiguity, aswell as to resolve any ambiguities within the sequence.

[0257] The Amino Acid Sequence of SIGLEC-BMS-L3a

[0258] The nucleotide sequence of Siglec-BMS-L3a (SEQ ID NO.:1, FIG. 2A,clone 526604), is predicted to represent a differentially spliced formof a Siglec-BMS-L3 transcript. The Siglec-BMS-L3a nucleotide sequenceencodes an open reading frame of 584 amino acids in length that exhibitsstructural properties shared by CD33. This nucleotide sequence encodesthe SIGLEC-BMS-L3a protein having the amino acid sequence described inSEQ ID NO.: 8 (FIG. 2B). SIGLEC-BMS-L3a includes an N-terminal 42 aminoacids hydrophobic signal peptide, a 397 amino acid extracellular domainincluding three Ig-like domains, a 25 amino acid residue transmembranedomain, and a 120 amino acid intracellular domain which includes twoputative ITIM motifs.

[0259] SIGLEC-BMS-L3a is expressed in eosinophils of an asthmaticpatient; therefore, SIGLEC-BMS-L3a may be a cell-surface receptor thatregulates adhesion and generates intracellular signals to directeosinophil maturation, recruitment, and activation in sites ofinflammation. Thus, SIGLEC-BMS-L3a may prove to be a potential targetfor asthma and other diseases of the immune system.

[0260] The Amino Acid Sequence of SIGLEC-BMS-L3b

[0261] The nucleotide sequence of Siglec-BMS-L3b, as described by SEQ IDNO.:2 (FIG. 3A, clone 527595), and represents a partial transcript thatis related to Siglec-BMS-L3a. The Siglec-BMS-L3b nucleotide sequenceencodes an ORF of 620 amino acids in length that exhibits structuralproperties shared by CD33 but lacks the first 17 amino acid residuescompared to the sequence of SIGLEC-BMS-L3a. This nucleotide sequenceencodes the SIGLEC-BMS-L3b protein having the amino acid sequencedescribed in SEQ ID NO.: 9 (FIG. 3B) that includes an incompleteN-terminal 15 amino acid hydrophobic signal peptide, a 475 amino acidextracellular domain including three Ig-like domains, an amino acidinsert sequence that is not found in SIGLEC-BMS-L3a, a 25 amino acidresidue transmembrane domain, and a 120 amino acid intracellular domainwhich includes two putative ITIM motifs.

[0262] The Amino Acid Sequence of SIGLEC-BMS-L3c

[0263] The nucleotide sequence of Siglec-BMS-L3c, as described by SEQ IDNO.:3 (FIG. 4A, clone 652995), represents a partial transcript that isrelated to Siglec-BMS-L3a. The Siglec-BMS-L3c nucleotide sequenceencodes an ORF of 573 amino acids in length that exhibits structuralproperties shared by CD33 but lacks the first 122 amino acid residuescompared to the sequence of SIGLEC-BMS-L3a. This nucleotide sequenceencodes the SIGLEC-BMS-L3c protein (SEQ ID NO.: 10, FIG. 4B) thatincludes an incomplete extracellular domain 428 amino acid residues inlength including three Ig-like domains, a 58 amino acid insert sequencethat is found in SIGLEC-BMS-L3d but not found in SIGLEC-BMS-L3b, a 25amino acid residue transmembrane domain, and a 120 amino acidintracellular domain which includes two putative ITIM motifs.

[0264] The Amino Acid Sequence of SIGLEC-BMS-L3d

[0265] The nucleotide sequence of Siglec-BMS-L3d, as described by SEQ IDNO.:4 (FIG. 5A, clone 1709963), represents a partial transcript that isrelated to Siglec-BMS-L3a The Siglec-BMS-L3d nucleotide sequence encodesan ORF of 431 amino acids in length that exhibits structural propertiesshared by CD33 but lacks the first 45 amino acid residues compared tothe sequence of SIGLEC-BMS-L3a, and lacks the sequences that encodes theC-terminal motifs. This nucleotide sequence encodes the SIGLEC-BMS-L3dprotein (SEQ ID NO.: 11, FIG. 5B) which is 410 amino acid residues inlength including, an incomplete extracellular domain, four Ig-likedomains, and a 20 amino acid residue transmembrane domain.

[0266] The Amino Acid Sequence of SIGLEC-BMS-L4a

[0267] The nucleotide sequence of Siglec-BMS-L4a, as described by SEQ IDNO.:5 (FIG. 7A, clone 2895823), represents a differentially spliced formof a Siglec-8 transcript. The Siglec-BMS-L4a nucleotide sequence encodesan open reading frame of 467 amino acids in length that exhibitsstructural properties shared by CD33 but lacks an unknown number ofN-terminal amino acid residues. This nucleotide sequence encodes theSIGLEC-BMS-L4a protein (SEQ ID NO.: 12, FIG. 7B) that includes, a 267amino acid extracellular domain including two Ig-like domains, a 24amino acid residue transmembrane domain, and a 30 amino acidintracellular domain which includes putative ITIM or ITAM motifs.

[0268] The Amino Acid Sequence of SIGLEC-BMS-L5a

[0269] The nucleotide sequence of Siglec-BMS-L5a, as described by SEQ IDNO.:6 (FIG. 8A, clone 3344926), represents a full-length cDNA clone of adifferentially spliced form of a Siglec-9 transcript. The Siglec-BMS-L5anucleotide sequence encodes an open reading frame of 464 amino acids inlength that exhibits structural properties shared by CD33. Thisnucleotide sequence encodes the SIGLEC-BMS-L5a protein (SEQ ID NO.: 13,FIG. 8B) that includes an N-terminal 15 amino acid hydrophobic signalpeptide, a 262 amino acid extracellular domain including two Ig-likedomains, a 24 amino acid residue transmembrane domain, and a 30 aminoacid intracellular domain which includes putative ITIM or ITAM motifs.

[0270] The Amino Acid Sequence of SIGLEC-BMS-L5b

[0271] The nucleotide sequence of Siglec-BMS-L5b, as described by SEQ IDNO.:7 (FIG. 9A, clone 3403156), represents a transcript that is relatedto Siglec-BMS-L5a, such as a differentially spliced form ofSiglec-BMS-L5a. The Siglec-BMS-L5b nucleotide sequence encodes an openreading frame of 287 amino acids in length that exhibits structuralproperties shared by CD33. This nucleotide sequence encodes theSIGLEC-BMS-L5b protein (SEQ ID NO.: 14, FIG. 9B) that includes anN-terminal 15 amino acid hydrophobic signal peptide, a 155 amino acidextracellular domain including only one Ig-like domain, and an inserthaving a sequence not found in SIGLEC-BMS-L5b which shifts the readingframe of the C-terminal end of this protein. The sequence ofSIGLEC-BMS-L5b lacks a transmembrane domain.

EXAMPLE 2

[0272] The following provides a description of analysis of theexpression patterns of Siglec-BMS transcripts in various human tissuesusing Northern blot techniques.

[0273] Northern blot membranes (FIG. 10A) were obtained from Clontech(MTN Blots, Clonetech, Palo Alto, Calif.). Each lane of the membranecontained approximately 1-2 micrograms of poly A+ RNA extracted fromvarious human tissues. Blots including RNA samples from human spleen,lymph node, thymus, PBL, bone marrow, and fetal liver (MTN Human ImmuneSystem II blot) and blots including RNA samples from human brain, heart,skeletal muscle, colon, thymus, spleen, kidney, liver, small intestine,placenta, lung, and PBL (MTN Human 12 lane blot) were each hybridizedwith probes generated by PCR methods, using full-length Siglec-BMS-L3 asa reference sequence beginning with the start codon ATG (FIG. 10B). TheL3 probe includes nucleotide sequences common in Siglec-BMS-3a, -3b,-3c, and -3d, from nucleotide position 596-1328. The SI probe includessplice variant sequences common in Siglec-BMS-3c and -3d from nucleotideposition 428-593. The S2 probe includes splice variant sequences commonin Siglec-BMS-3b and -3c from nucleotide position 1341-1578. All threeprobes were amplified from the Siglec-BMS-L3c sequence (e.g., 652995).Additionally, a β-actin probe was used as a control probe (Clontech,Palo Alto, Calif.).

[0274] PCR primers used to generate the probes for the Northern analysisincluded: L3: 5′ (596-616) TGC TCA GCT TCA CGC CCA GAC (SEQ ID NO:33)3′ (1319-1328) TGC ACG GAG AGG CTG AGA GA (SEQ ID NO:34) Probe length:732 bp S1: 5′ (428-446) CTC AGA AGC CTG ATG TCT A (SEQ ID NO:35)3′ (576-593) GAG AAG TGG GAG GTC GTT (SEQ ID NO:36) Probe length: 65 bpS2: 5′ (1341-1359) CTG CTG GGC CCC TCC TGC (SEQ ID NO:37) 3′ (1559-1578)GAC GTT CCA GGC CTC ACA G (SEQ ID NO:38) Probe length: 237 bp

[0275] Reference Sequence: Full Length BMSL3 starting with ATG

[0276] The probes were individually labeled with ³²P-dCTP by randompriming, purified on a Chromospin 100 column (Clontech), andheat-denatured. The membranes were pre-hybridized in ExpressHyb Solution(Clontech) at 68 degrees C. for 30 minutes with continuous shaking. Themembrane was incubated with the denatured probes (approximately 2million cpm per ml) in fresh ExpressHyb Solution for 4 hours at 68degrees C., with continuous shaking. The membrane was washed in severalchanges of 2× SSC, containing 0.05% SDS, for 40 minutes at roomtemperature. The wash was followed by several changes of 0.1× SSC,containing 0.1% SDS, for 40 minutes at 50 degrees C. The hybridizationpattern of the membranes was obtained using Phosphorlmager 445 SI(Molecular Dynamics, Sunnyvale, Calif.).

[0277] The L3 probe readily detected a 4.4 kb transcript in human immunetissues, including spleen, lymph node, and PBL. Lower levels ofSiglec-BMS-L3 transcripts were also detectable in human, thymus, bonemarrow, and fetal liver (FIG. 10A). The Siglec-BMS-L3 transcripts werenot detected in human non-immune tissues including brain, heart,skeletal muscle, colon, kidney, liver, small intestine, placenta orlung.

EXAMPLE 3

[0278] The following provides a description of the analysis of theexpression patterns of Siglec-BMS transcripts in various human tissuesusing standard reverse transcriptase PCR amplification techniques.

[0279] Reverse transcriptase PCR methods were employed to determine thetissue distribution of Siglec-BMS transcripts in RNA extracted fromprimary human cells and cell lines and commercially available humanorgan cDNA. Human I, II and Immune Multiple Tissue cDNA Panels werepurchased from Clontech. In addition, RNA was extracted from monocytes,TNF-stimulated endothelial cells, spleen, HL-60 cells and Jurkat cellswith Trizol (Gibco BRL, Grand Island, N.Y.) according to the directionsof the manufacturer.

[0280] The extracted RNA was reverse transcribed into cDNA using thefollowing reaction mixture: 5 micro grams total RNA from each sample in8 micro liters diethyl cyanophosphate-treated (DEPC) water, 4 microliters 5x first strand buffer (Gibco BRL), 2 micro liters 10 mMdeoxynucleotide triphosphate (dNTP) (Gibco BRL), 2 micro liters 0.1 MDTT (Gibco BRL), 1 micro liters RNAse inhibitor (40 U, Roche MolecularBiochemicals, Indianapolis, Ind.), 2 micro liters 10× hexanucleotides(Roche Molecular Biochemicals) and 1 micro liter SuperScript II (GibcoBRL). The reaction mixture was incubated at 37 degrees C. for 1 hour,then at 75 degrees C. for 15 minutes, and stored at 4 degrees C. Customprimers were obtained from Life Technologies (Gaithersburg, Md.) andsequencing parameters optimized for each primer pair. The quality of thePCR products was determined by electrophoresis on a 1.2% agarose gel.PCR was carried out using “Ready-To-Go” PCR Beads (Pharmacia BiotechInc., Piscataway, N.J.) in a 25 micro liters reaction mixture including:1.5 U Taq polymerase, 10 mM Tris-HCl (pH 9.0 at room temperature), 50 mMKCl, 1.5 mM MgCl₂, 200 μM of each dNTP and stabilizers, including BSA,0.2 micro M of each primer, and 1 micro liter of RT-PCR reaction productor 2 ul of each of the commercially prepared MTC Human I, II and ImmunecDNA panels from Clontech.

[0281] PCR primer sequences used for the PCR reactions included: P1primers: 5′ (−96/−77) CCT TCG GCT TCC CCT TCT GC (SEQ ID NO:39)3′ (560-579) CGT TGG TTT GGT TCC TTG G (SEQ ID NO:40) P2 primers:5′ (852-870) CAC ACT GAG CTG GGT CCT G (SEQ ID NO:41) 3′ (1560-1578) GACGTT CCA GGC CTC ACA G (SEQ ID NO:42) P3 primers: 5′ (852-870) CAC ACTGAG CTG GGT CCT G (SEQ ID NO:43) 3′ (1670-1689) GAA AAG AAG AGC CGT GATGC (SEQ ID NO:44)

[0282] Expected product size for each L3 splice variant BMS-L3 P1primers P2 primers P3 primers A:526604 None None 547 bp B:527595 None727 bp 838 bp C:652995 675 bp 727 bp 838 bp D:1709963 675 bp None 547 bp

[0283] P3 primers were expected to be 552 bp, 837 bp, 837 bp and 552 bpin length, as shown in the table above.

[0284] The results are shown in the table depicted in FIG. 11A.

EXAMPLE 4

[0285] The following provides a description of the analysis of theexpression patterns of Siglec-BMS transcripts in various human tissuesblood cells and cell lines, using SYBR Green PCR amplificationtechniques.

[0286] Human I, II and Immune Multiple Tissue cDNA Panels were purchasedfrom Clontech (Palo Alto, Calif.). In addition, RNA was obtained fromlymphocytes, eosinophils, neutrophils, T-cells, monocytes,TNF-stimulated endothelial cells, spleen cells, HL60 cells and Jurkatcells and reverse transcribed as described in Example 3 above. The cDNAwas amplified using the SYBR Green PCR Master Mix (PE Biosystems, FosterCity, Calif.). The SYBR Green system permits relative quantification ofa target transcript sequence compared to an internal house-keeping gene,beta-actin, with real-time monitoring of the amplification (PEBiosystems, User Bulletin #2 P/N 4303859). The reaction was performed onan ABI PRISM 7700 Sequence Detection System (PE Biosystems). Allamplifications were normalized for beta-actin gene in the linear portionof the amplification curves.

[0287] The following primer pairs were used for amplification ofdifferent regions of the L3 gene (FIG. 12A): the L3-TM primer pairincludes the putative transmembrane sequences common amongSiglec-BMS-3a, -3b, -3c, and -3d, from nucleotide position 1603 to 1966;the S1 primer pair includes splice variant sequences common amongSiglec-BMS-3c and -3d from nucleotide position 428 to 447; and the S2primer pair includes splice variant sequences common among Siglec-BMS-3band -3c from nucleotide position 1948 to 1966. The data was normalizedto beta-actin gene expression and then expressed as fold-increase overskeletal muscle, which served as a reference tissue (FIG. 12B).

[0288] PCR primers for the SYBR Green amplification methods included:L3-TM: 5′ (1603-1621) TGC AGC TGC CAG ATA AGA (SEQ ID NO:45)3′ (1948-1966) GGC TTG AGT GGA TGA TTT (SEQ ID NO:46) PCR product: 363bp S1: 5′ (428-447) CTC CGA AGC CTG ATG TCT A (SEQ ID NO:47)3′ (576-594) GAG AAG TGG GAG GTC GTT (SEQ ID NO:48) PCR product: 166 bpS2: 5′ (1343-1361) CTG CTG GGC CCC TCC TGC (SEQ ID NO:49) 3′ (1560-1579)GAC GTT CCA GGC CTC ACA G (SEQ ID NO:50) PCR product: 236 bp Beta-actin:5′ GTG GGG CGC CCC AGG CAC CA (SEQ ID NO:51) 3′ CTC CTT AAT GTC ACG CACGAT TC (SEQ ID NO:52) PCR product: 539 bp

EXAMPLE 5

[0289] The following provides a description of the mapping of the humanchromosomal location of Siglec-BMS-L3, that generated the variousdifferentially spliced transcripts, including Siglec, BMS-L3a, -L3b,-L3c, and -L3d.

[0290] The human chromosomal map location of Siglec-BMSL3 was determinedusing the Stanford G3 radiation hybrid panel (Stanford University GenomeCenter Radiation Hybrid Mapping Server). A primer pair was chosen thatwould allow amplification of a 150 bp fragment from the transmembraneregion. The PCR conditions included: 95 degrees C. for 5 minutes;followed by 30 cycles of 95 degrees C., 56 degrees C., 72 degrees C.,for 30 seconds each; followed by 72 degrees C. for 10 minutes.

[0291] The primers were used to screen all 83 hybrids of the Stanford G3set. The resulting pattern of positives and negatives was submitted tothe Stanford Human Genome Center Radiation Hybrid Mapping Server, whereit was subjected to a two-point statistical analysis against 15,632reference markers. This analysis yielded a linkage to two markers,D19S425 and D19S418 at a distance of 32 cR [Log of Odds (LOD)score=6.47] and 29cR (LOD score=6.28), respectively, and corresponded toan approximate physical distance of 960 and 870 kb, respectively, inthis panel (1 cR=30 kb). Reference to the Stanford Radiation Hybrid Mapof this region of chromosome 19 gives the most likely order ofD19S418-Siglec BMSL3-D19S425, with a cytogenetic location of 19q13. Themarker D19S418 is positive with YAC 790A05 of the Whitehead genetic mapof Chromosome 19 (Wende et al. Mammal Gen., 10, 154-160 (1999)).

[0292] PCR primers used for the chromosomal location methods included:L3-TM: 5′ (1603-1621) TGC AGC TGC CAG ATA AGA (SEQ ID NO:53)3′ (1948-1966) GGC TTG AGT GGA TGA TTT (SEQ ID NO:54) PCR product: 363bp

EXAMPLE 6

[0293] The following provides a description of the generation of Igfusion proteins comprising the extracellular domains of Siglec-BMS-L3aand Siglec-BMS-L3-995-2 fused to the human R gamma chain.

[0294] Plasmids encoding the Ig fusion proteins were constructed.Briefly, nucleotide sequences encoding the extracellular domains ofeither SIGLEC-BMS-L3a (e.g., 526604) or SIGLEC-BMS-L3-995-2 wereamplified from a liver cDNA library (Clontech) by PCR methods. Thenucleotide sequence encoding the extracellular domain of SIGLEC-BMS wasoperatively ligated into a proprietary expression vector, pd19(Bristol-Myers Squibb, Princeton, N.J.). The pd19 vector has acytomegalovirus promoter (CMV promoter; Boshart, M. et al., 1985 Cell41:521-530) to drive expression of Siglec-BMS-L3a andSiglec-BMS-L3-995-2 sequences. Additionally, the pd19 vector includes aportion of the human R gamma chain having a point mutation which reducesFc receptor binding of the immunoglobulin portion encoded therein. Theresulting plasmids were designated SiglecL3A-hIg and SiglecL3-hIg. TheSiglecL3-hIg fusion proteins were expressed in COS cells byDEAE-transient transfection. The fusion protein was purified from COS7supernatant by chromatography using Protein A trisacryl column (Pierce,Rockford, Ill.). before use.

EXAMPLE 7

[0295] The following provides a description of the determination of thebinding specificity of the extracellular domain of SIGLEC-BMS-L3A andSIGLEC-BMS-L3 fusion proteins, using FACs analysis.

[0296] Mixed white blood cell populations and hemopoietic cell lineswere obtained to determine the binding specificities of SIGLEC-BMS-L3Aand SIGLEC-BMS-L3. The cells and cell lines used in this analysisincluded the following: cell lines MB, PM, and TJ which are EBVtransformed B-cells (Bristol-Myers Squibb); B-cell lymphoblastomasRamos, HSB-2, and Raji; Jurkat which is a T-cell lymphoblastoma; HELwhich is a erythroblastic leukemia cell line HEL; and monocytic celllines U973 and HL60 which were obtained from the American Type CultureCollection (Manassas, Va.).

[0297] The cells were suspended in binding buffer (DMEM including 1% w/vbovine serum albumin and 0.1% sodium azide), with the Siglec fusionprotein (Example 6), mALCAM hIg fusion protein (R-gamma fusion proteincontrol), or CD5 hIg fusion protein (E-gamma fusion protein control) ata concentration of 5 micro grams of protein/1×10⁶ cells. Rabbit Ig(Sigma Chemical Co., St. Louis, Mo.) was also added at 100 micrograms/million cells to prevent non-specific binding of the Ig tail onthe fusion proteins to the Fc receptors. The mixture was incubated onice for 1 hour followed by two washings with binding buffer. The cellswere centrifuged at 500× G for 5 minutes between each wash.Anti-hIg/FITC (Jackson Immunoresearch, West Grove, Pa.) and/orphycoerythrin-conjugated anti-CD20/PE (Beckton Dickenson, San Jose,Calif.), anti-CD14/PE (Beckton-Dickenson), and anti-CD4/PE(Beckton-Dickenson) were added on ice for 30 minutes. After furtherwashing, the cells were analyzed on a Becton Dickenson FACSort usingCell Quest software. Cells were live gated and red/green color wascompensated.

[0298] The mixed white blood cell populations were analyzed for bindingto the SIGLEC-BMS fusion proteins. Results of FACs analysis are shown inTable 1. There was no difference in the binding of SIGLEC BMSL3a andSIGLEC BMSL3 to the cells that were examined by FACs. A small populationof lymphocyte-sized cells and monocyte-sized cells stained positivelyfor both fusion proteins. Double staining with either anti-CD20 (forB-cells), anti-CD-14 for monocytes, anti-CD4 or anti-CD3 (for T-cells)determined that B-cells and monocytes were binding the fusion protein,but T-cells were not. Possible binding of the fusion protein to the Fcreceptors on B-cells and monocytes was ruled out by comparison with twofusion protein controls, one with a similar R-gamma hIg tail thatdoesn't bind FcR (mALCAM hIg) and one with an E-gamma hIg tail that doesbind FcR (CD5 hIg).

[0299] Similar FACs analyses were performed with cell lines. The HEL(e.g., an erythroblastic leukemia cell line) and Jurkat (e.g., a T-cellline) cell lines did not stain positively for either SIGLEC BMSL3 fusionprotein. Additionally, the EBV-transformed B cell lines MB, PM and TJdid not stain positively. The B-cell lines, Ramos, Raji and HSB2, didstain positively. Although some monocyte binding was observed in wholeblood, the monocytic cell lines, U973 and HL60, did not exhibit anybinding.

[0300] Table 1 is a FACs analysis of Siglec-10-hIg binding. Data wasobtained by incubating mixed white blood cell populations andhemapoietic cell lines with Siglec-10-hIg fusion protein then stainedwith fluorescein-conjugated anti-hIg (Jackson Immunoresearch, WestGrove, Pa.) and/or phycoerythrin-conjugated anti-CD20, anti-CD3,anti-CD14, and anti-CD4. mALCAM hIg fusion protein (hIg Rγ control) andCD5 hIg fusion protein (hIg E7 control) were analyzed in parallel ascontrols. Rabbit Ig (Sigma) was also added to prevent non-specificbinding of the Ig tail on the fusion proteins to Fc receptors. Thepercentage of cells staining positive for FITC compared to backgroundwith mALCAM, CD5 and Siglec-10 hIg is shown. One color FACs was used forcell lines and two color FACs was used for primary peripheral bloodmononuclear cells (PBMC). TABLE 1 FITC Staining (%) mALCAM CD5 Cell lineType hIg hIg Siglec-10-hIg MB B-cell (EBV) 0 0 0 PM B-cell (EBV) 0 0 0TJ B-cell (EBV) 0 0 0 Ramos B-cell (lymphoma) 0 4 57 HSB-2 B-cell(lymphoma) 0 0 24 Raji B-cell (lymphoma) 1 2 36 Daudi B-cell (lymphoma)0 0 20 Jurkat T-cell (lymphoma) 38 2 0 HEL RBC (leukemia) 0 0 0 U973Monocyte (leukemia) 0 4 0 HL60 Monocyte (leukemia) 0 0 0 FITC Staining(%) Blood ALCAM CD5 Population PE+ (%) hIg hIg Siglec-10-hIg PBMC CD20+ 7 0 0 4 CD14+ 11 0 0 8 CD4lo+ 12 0 0 11 CD4hi+ 63 8 0 0 CD3+ 65 4 0 0Granulocytes 0 0 0

EXAMPLE 8

[0301] The following provides a description of the determination ofwhether distinct blood cell populations or cell lines exhibit bindingspecificity for the extracellular domain of SIGLEC-BMS-L3 fusionprotein, using a solid support method.

[0302] The SIGLEC-BMS-L3 fusion protein was immobilized on a solidsupport, by coating an ELISA plate with SIGLEC-BMSL3 hIg fusion protein(200 ng/well) overnight. The plate was blocked for 1 hour with DMEMcontaining 1% BSA.

[0303] The cells and cell lines used included: mixed white blood cells,mixed granulocytes, purified B-cells, purified NK cells, purifiedmonocytes, and Ramos (B-cell line), RBCs, Jurkats (T-cell line), andHL60s and K652 (monocytic cell lines). The blood cells and cell lineswere labeled with calcein-AM (5 micro liter/10⁸ cells) for 30 minutes at37 degrees C. The cells were washed two times in Hanks buffered saltsolution (HBSS) and added to the blocked ELISA plates (4×10⁵/well in 200micro liters) at 37 degrees C. for 30 minutes. The plates were gentlywashed with HBSS and 100 micro liters HBSS was added to each well.Fluorescence was read on a CytoFluor 4000 (PerSeptive Biosystems,Framingham, Mass.) at 485 excitation/530 emission.

[0304] The results are shown in FIG. 13. The mixed white blood cells,mixed granulocytes, purified B-cells, purified NK cells, purifiedmonocytes, and Ramos (B-cell line) adhered to the immobilized SIGLECfusion protein. RBCs, Jurkats (T-cell line), HL60s and K652 (monocyticcell lines) did not adhere to the protein-coated plate. Sialidasepretreatment of the cells (0.1 U/ml for 30 minutes at 37 degrees C.) didnot significantly affect binding of any of the adherent cell types.

EXAMPLE 9

[0305] The following provides a description of the binding studies usingCOS cells expressing the full length SIGLEC-BMS-L3 (e.g., 995-2, seeExample 14) protein and various cells and cell lines.

[0306] COS7 cells were transiently transfected or mock-transfected witha pcDNA3 plasmid (InVitrogen, Carlsbad, Calif.) containing a full lengthSiglec-BMSL3 (e.g., 995-2, see Example 14) by the DEAE-dextran method.Twenty four hours after transfection, the cells were lifted from theplates with EDTA and re-plated in 6-well plates containing DMEM with 10%FCS at a density of 2×10⁵/well. Binding assays were performed between 48and 60 hours post-transfection.

[0307] Blood cells and cell lines were labeled with calcein-AM (5 microliters/10⁸ cells) for 30 minutes at 37 degrees C. RBCs, mixed whiteblood cells, Ramos (B-cell line), HL60 and K562 (monocytic cell lines),and Jurkats (T-cell line) were suspended in DMEM containing 0.25% BSA.Some cells were also pre-treated with sialidase (0.1 U/ml for 30 minutesat 37 degrees C. followed by 3 washes with DMEM +0.25% BSA). One ml ofblood cells or a cell line suspension was added to each well. The cellswere incubated together at 37 degrees C. for 30 minutes with gentlerocking. The plates were washed gently 3 times with PBS +0.25% BSA. Thecells were fixed with 0.25% glutaraldehyde.

[0308] To quantify binding, the percentage of transfected COS7 cellsthat bound two or more of the added cell types was determined from 10fields in each treatment (at least 100 cells from each treatment werescored). The results were expressed as a percentage of COS7 cellbinding. Binding to the transfected cells was also compared to themock-transfected controls.

[0309] The results are shown in FIG. 14. The transfected COS7 cellsbound to the mixed white blood cells and Ramos cell line (B-cell line).This binding was not significantly affected by sialidase pretreatment.Since there was no indication that the sialic acid digestion wascomplete, this observation is only suggestive. The transfected COS7cells did not exhibit binding to the RBCs, Jurkats (T-cell line), or toHL60 and K562 (monocytic cell lines).

EXAMPLE 10

[0310] The following provides a description of the generation of variousfusion proteins comprising the cytoplasmic tail domain of Siglec-BMS-L3afused to the GST protein.

[0311] To construct a nucleotide sequence encoding the fusion proteincomprising the cytoplasmic tail domain of the SIGLEC-BMS-L3 protein, thecytoplasmic domain of SIGLEC-BMS-L3 was amplified from a PHA-activatedJurkat cDNA library (KRRTQTE . . . VKFQ*; e.g., see FIG. 6B). Thecytoplasmic tail fragment was subcloned, via EcoRI/XhoI sites, intopGEX4T-3 (Pharmacia Biotech) which includes the GST sequenceTheresulting construct was designated GST-SiglecL3cyto (FIG. 15).

[0312] In addition, Y→F mutants were generated at positions 597, 641,and 691. GST-SiglecBMSL3cyto and the SIGLEC-BMS mutant proteins wereexpressed in E. coli bacteria and purified according to a Pharmaciaprotocol (based on the methods of Smith and Johnson, 1988 Gene67:31-40).

[0313] PCR primers used to generate the sequence encoding thecytoplasmic tail domain of SIGLEC-BMS-L3cyto(wildtype) and the mutantSIGLECs included the following: GST-SiglecBMSL3cyto (wt) primers: 5′ GCGGCC AGG AAT TCC AAG AGA CGG ACT CAG ACA GAA (SEQ ID NO:55) 3′ GCG GCCCTC GAG TCA TTG GAA CTT GACTTC TGC (SEQ ID NO:56) GST-Sig1ecBMSL3Y641F:wt forward and reverse primers and Y641F mutagenic primers: 5′ CCA GAATCA AAG AAG AAC CAG AAA AAG GAG TTT GAG TTG CCC AGT TTC CCA GAA CCC (SEQID NO:57) 3′ GGG TTC TGG GAA ACT GGG CAA CTG AA CTG CTT TTT CTG GTT CTTCTT TGA TTC TGG (SEQ ID NO:58) GST-SiglecBMSL3Y667F: wt forward andreverse primers and Y667F mutagenic primers: 5′ GAG AGC CAA GAG GAG CTCCAT TTT GCC ACG CTC AAC TTC CCA GGC (SEQ ID NO:59) 3′ GCC TGG GAA GTTGAG CGT GGC AAA ATG GAG CTC CTC TTG GCT CTC (SEQ ID NO:60)GST-SiglecBMSL3Y691F: wt forward and Y691F mutagenic reverse primers:5′ GCG GCC CTC GAG TCA TTG GAA CTT GAC TTC TGC AAA ATC CGC CTG GGT GCC(SEQ ID NO:61) 3′ GCG GCC CTC GAG TCA TTG GAA CTT GAC TTC TGC AAA ATCCGC CTG GGT GCC (SEQ ID NO:62) GST-SiglecBMSL3Y641 alone (deletionmutant) primers: 5′ GCG GCC AGG AAT TCC ATC AAT GTG GTC CCG ACG GCT GGC(SEQ ID NO:63) 3′ GCG GCC CTC GAG TCA ATG GAG CTC CTC TTG GCT CTC (SEQID NO:64)

EXAMPLE 11

[0314] The following provides a description of the generation of thevarious fusion proteins comprising the extracellular domain ofSiglec-BMS-L3a or Siglec-BMS-L3 fused to human Ig sequences. The fusionproteins include Siglec-BMS-L3a hIg and Siglec-BMS-L3 hIg.

[0315] The nucleotide sequences encoding the extracellular domain ofSIGLECBMS-L3a (e.g. 526604) and 995-2 were amplified using primerscontaining linker sequences with restriction sites for Hind III, Bgl IIand NcoI. The amplified fragments (e.g., 1201 bp fragment forsiglecBMS-L3a or 1650 bp fragment for Siglec BMS-L3) were digested withrestriction enzymes Hind III and Bgl II, and the digested fragments werecloned into a pd19 vector (see Example 6; Bristol-Myers Squibb,Princeton, NJ) which was digested with Hind III and BamHI. The pd19vector includes a portion of the human R gamma chain having a pointmutation which reduces Fc receptor binding of the immunoglobulin portionof the encoded fusion protein. The integrity of the insertions wasvalidated by digesting the Siglec/hIg plasmid constructs with eitherHind III/Nco I to check the extracellular domain of Siglec or with HindIII/Xba I to check the entire fusion construct. The Siglec-10-hIg fusionprotein was expressed in COS7 cells by DEAE-dextran transienttransfection. COS7 cells were transfected with I micro gram/milli literDNA in CMEM containing 1% DEAE-dextran (Sigma), 0.125% chloroquine(Sigma) and 10% NuSerum (Beckton Dickenson, Franklin Lakes, N.J.) for 4hours followed by two minute treatment with 10% DMSO in phosphatebuffered saline (PBS). After 4-7 days, the COS7 supernatant was removedand Siglec-10-hIg fusion protein was purified by chromatography over aprotein A trisacryl column (Pierce, Rockford, Ill.).

[0316] Sequence of the primers used to construct Siglec-BMSL-3a hIgincluded the following:            Hind III 5′ CCG CCT AAG CTT  TCC CCTTCT GCC AAG AGC CCT GAG CCC TGA GCC (SEQ ID NO:65) ACT CAC AGC ACG ACCAGA GAA CAG GCC TGT CTC AGG CAG GCC CTG CGC CTC CTA TGC GGA GAT G       Bgl II         Nco I 3′ GA A GAT CT G AA C CAT G GT TAT AGT GCACGG AGA GG (SEQ ID NO:66) Sequence of the primers used to constructSiglec-BMS-L3 hlg included the following:            Hind III 5′ CCGCCT AAG CTT  TCC CCT TCT GCC AAG AGC CCT GAG CCC TGA GCC (SEQ ID NO:67)ACT CAC AGC ACG ACC AGA GAA CAG GCC TGT CTC AGG CAG GCC CTG CGC CTC CTATGC GGA GAT G      Bgl II     Nco I 3′ GA A GAT CT G AA C CAT G GT TAGGAG AAT GCC GTT GA (SEQ ID NO:68)

EXAMPLE 12

[0317] The following provides a description of kinase assays used todetermine if the cytoplasmic tail domain of SIGLEC-BMS-L3 undergoesphosphorylation by known tyrosine kinases.

[0318] The kinase assays were run in an ELISA format usingrepresentatives of the four major tyrosine kinases known to associatewith receptors similar in nature to SIGLEC-BMS-L3. The tyrosine kinasestested included: lck, ZAP70, emt, and JAK3.

[0319] The GST fusion proteins and GST were coated on Immulon 2 96-wellplates at 4 micro grams/ml in sodium carbonate pH 9 for 16 hours at roomtemperature. The GST fusion proteins included: GST-SiglecBMSL3cyto(wildtype), GST-LAT (an adapter protein with 10 tyrosines available forphosphorylation), GST-cyto-Y597F (Y→F mutation at the 597 position),GST-L3cyto-Y641F (Y→F mutation at the 641 position), GST-L3cyto-Y667F(Y→F mutation at the 667 position), L3cyto-Y691F (Y→F mutation at the691 position), GST-L3cyto-Y641alone, and GST alone. LAT is a 36-38 kDapalmitoylated, integral membrane adapter protein expressed in T-cells,mast cells, NK cells, and megakaryocytes. Signal transduction throughthe T-cell receptor (TCR/CD3) involves the activation of tyrosinekinases and the subsequent phosphorylation of numerous cytoplasmicprotein substrates. LAT has 10 tyrosine residues and is one of the majorsubstrates of these many families of tyrosine kinases. PhosphorylatedLAT is a good positive control because it binds many critical signalingmolecules (W. Zhang, et al., 1998 Cell 92: 83; W. Zhang, et al., 1999Immunity 10: 323).

[0320] The plates were washed and then blocked with blocking reagent(Hitachi Genetics Systems, Alameda, Calif. -). The kinase reactions wereconducted in 50 microliter volumes, in kinase buffer (25 mM Hepes pH7.0, 6.25 mM MnCl₂, 6.25 mM MgCl₂, 0.5 mM sodium vanadate, 7.5 micro MATP) and two fold dilutions of the tyrosine kinases starting at aconcentration of 0.25 micro grams/ml. The kinase reactions wereincubated for 1 hour at room temperature. The plates were washed and thephospho-tyrosine content was detected with anti p-Tyr (PY99) HRP (SantaCruz, Santa Cruz, CA) at 1:1000 and peroxidase substrate (KPL,Gaithersburg, Md.). Absorbance was detected at 650/450 nm.

[0321] The results—of the Kinase assays shown in FIGS. 16A through Gindicate that the cytoplasmic domain of the SIGLEC-BMS-L3 protein can bephosphorylated by representatives of at least three of four majorfamilies of kinases: Jak3, Lck, Emt but not ZAP-70. By titering thekinase concentration, it was determined that Siglec-10 could bephosphorylated equally well by Lck and Jak, moderate phosphorylation wasobserved with Emt and little or no phosphorylation occurred with ZAP-70.The wildtype GST-SIGLECBMS-L3cyto was phosphorylated by Lck(100%)>JAK3(92%)>>emt(65%)>>>ZAP70 (20%).

[0322] The GST fusion proteins having mutations at particular positionsin the cytoplasmic tail affected phosphorylation by the various tyrosinekinases. The results are summarized in the following table. Decrease inWild type Phosphorylation Mutant lck JAK3 emt ZAP70 Y641F — — — — Y667F50% 50% 70% 100% Y691F 30% 25% — — Y641 alone — — —  20%

[0323] The results shown in FIGS. 16A through G and in the table abovesuggest that the tyrosines at positions 597 and 667, contained within anITIM-like motif, are likely targets of phosphorylation by severalclasses of tyrosine kinase signaling molecules, including lck, JAK3, emtand ZAP70. The tyrosine located at position 691 was also contributing tothe phosphorylation of wild type Siglec tail by Lck and Jak3 kinases.For example, phosphorylation of the Y's at positions 667 and 691accounted for approximately 80% of the wildtype phosphorylation by lckand 75% of the wildtype phosphorylation by JAK3. By comparison, themutation of the Y at position 641 did not significantly affect thedegree of phosphorylation by any of the kinases that were tested. Inaddition, a construct containing Y641 alone was not phosphorylated byany of the kinases, confirming that Y641 is most likely not a site forphosphorylation (data not shown). The contribution of the Y at position597 to phosphorylation, could be calculated to be approximately 20% forlck, 25% for JAK3 and 30% for emt.

EXAMPLE 13

[0324] The following provides a description of the use of Westernblotting and ELISA techniques to determine if the cytoplasmic taildomain of SIGLEC-BMS-L3 binds SHP-1 or SHP-2 in cell lysates.

[0325] Western Blotting For SHP proteins:

[0326] To determine if the cytoplasmic domain of Siglec-10 binds SHP-1and SHP-2 in cell lysates, 10 μg of GST fusion protein +/−tyrosinephosphorylation were incubated with 300 μl of cell lysate(Triton-X-100-soluble fraction 5×10⁷ unstimulated cells) at 4° C.overnight. The GST fusion protein complexes were captured with 50 μl ofglutathione-sepharose beads (Amersham Pharmacia Biotech) for 1 hr at 4°C. The beads were then incubated with Jurkat cell lysates. The beadswere washed three times with ice cold lysis buffer, and bound proteinswere eluted in SDS reducing sample buffer and resolved byelectrophoresis on an SDS-polyacrylamide gel. The separated proteinswere transferred to nitrocellulose by standard western blottingtechniques. The blots were then stained for proteins containingphosphorylated tyrosines using anti-P-Y HRP-conjugated antibody (Clone4G10, Upstate Biotechnology, Lake Placid, N.Y.). Blots were thenstripped and stained with either mouse anti-SHP-1 (TransductionLaboratories, Lexington, Ky.) or mouse anti-SHP-2 (TransductionLaboratories) followed by an HRP-conjugated secondary antibody (goatanti-mouse, Biosource Int., Camarillo, Calif.) Stained proteins wereimaged by adding a chemiluminescent detection reagent (Renaissance, NENBio Products, Boston, Mass.) and exposing to film (Kodak).

[0327] The results, shown in FIG. 17A, indicate that both SHP-1 andSHP-2 from the cell lysates are capable of binding to the cytoplasmicdomain—of the SIGLEC L3 protein. The binding of SHP-1, however, wasmissing from the Y667F mutant, indicating this to be the preferredtyrosine (e.g., single-letter code:Y) for interaction with SHP-1. SHP-2binding, however, was only diminished by about 50% in the Y667F mutantsample, indicating that SHP-2 may be binding both to the tyrosine atposition 667 and to other tyrosines in the cytoplasmic tail of SIGLECBMSL3.

[0328] ITIM Peptide Binding to SHP Proteins by ELISA:

[0329] A biotinylated Siglec-10 phosphopeptide (660-678)ESQEELHpYATLNFPGRVPR (ITIM667) was produced by W.M.Keck BiotechnologyResource Center, New Haven Conn. Four μg/ml of phosphopeptide inBlocking Reagent (Hitachi Genetics Systems) was bound to astrepavidin-coated ELISA plate (Pierce, Rockford, Ill.). Plates werewashed and then two fold dilutions of the GST fusion proteins, GSTalone, GST-SHP-1SH2SH2 or GST-SHP-2SH2SH2 or GST-ZAP-70SH2SH2 were addedand incubated for 1 hour at room temperature. Polyclonal anti-GST(prepared in-house by procedures similar to those detailed for Siglecantibody production) was added at 1:1000, HRP-conjugated anti-Rabbit(Biosource at 1:2000 was added and signal detected with peroxidasesubstrate (KPL, Gaithersburg, Md.).

[0330] The results of the cell-free system shown in FIG. 17B, alsoconfirmed that SHP-1 and SHP-2 could both bind with high affinity to aphosphorylated peptide containing the Y667 domain.

EXAMPLE 14

[0331] The following provides a description of the generation of a DNAmolecule having the sequence of full-length Siglec-BMS-L3, which encodesthe SIGLEC-BMS-L3 protein. The full-length sequence was designatedBMSL3-995-2 (FIGS. 6A and B).

[0332] The clone designated 652995 (Incyte database), fused to a pSPORTvector (Life Technology/Gibco, Grand Island, N.Y.) includes a complete3′ end of the Siglec BMS-L3 cDNA. The 652995 clone was digested withrestriction enzymes EcoRI and BbrPI and the larger fragment(approximately 6.4 kb) was gel-purified. A second clone, designated3421048 (Incyte database), included a complete 5′ end of the SiglecBMS-L3 cDNA and was digested with restriction enzymes EcoRI and BbrPIand gel purified (approximately 820 bp). The gel purified fragments wereligated into a pSPORT vector, resulting in a hybrid construct havingfull-length Siglec BMS-L3 nucleotide sequences and was designated 995-2.The sequence of 995-2 was verified against other SiglecBMS-L3 sequences.The 995-2 clone was digested with restriction enzymes EcoRI and Not I,and ligated into a similarly digested pcDNA3 vector (Invitrogen,Carlsbad, Calif.) for full length expression.

[0333] A partial sequence of the 5′ end of the 3421048 was obtained. Thesequence is as follows: 5′CAGGCCTGTC TCACGCAGGC CCTGCGCCTC CTATGCGGAGATGCTACTGC (SEQ ID NO:69)   CACTGCTGCT GTCCTCGCTG CTGGGCGGGT CCCANGCTATGGATGGGAGA   TTCTGGATAC GAGTGCAGGA GTCAGTGATG GTGCCGGAGG GCCTGTGCAT  CTCTGTGCCC TGCTCTTTCT CCTACCCCCG ACAGGACTGG ACAGGGTCTA   CCCCAGCTTATGGCTACTGG TTCAAAGCAG TGACTGAGAC A3′

EXAMPLE 15

[0334] The following Example provides a description of PolyacrylamideGlycoconjugate Binding Assays to analyze binding of Siglec-10 to sialicacid.

[0335] COS7 cells were transiently transfected (see methods above fortransfection protocol) with full length Siglec-10 (995-2 in pcDNA3vector) or sham transfected were plated in 96-well plates within 24hours of transfection and allowed to attach for 18-22 hours. Half of theplated cells were treated with 0.01 U sialidase (Calbiochem, La Jolla,Calif.) for 1 hour at 37° C. because the treatment has been shown toremove cell surface sialic acids that possibly mask the binding site forother Siglec family members (Zhang et al., 2000). The cells were thenwashed with DMEM containing 1%BSA and incubated with saturatingconcentrations (20 μg/ml) of a polyacrylamide polymer containing biotinand carbohydrate (lactose, 3′-sialyllactose or 6′ sialyllactose,GlycoTech Corp., Rockville, Md.). In a parallel cell-free experiment,Immulong plates were coated with purified Siglec-10-hIg fusion protein(200 ng/well) and incubated with 20 μg/ml of the polyacrylamidepolymers. After 1 hour, plates were washed and treated withstreptavidin-horse radish peroxidase (Vector Labs, Burlingame, Calif.)in DMEM for 30 minutes. After a final wash, TMB peroxidase substrate(KPL, Gaithersburg, Md.) was added and the plates were developed at roomtemperature. The reaction was stopped with 0.1N HCl and absorbance at450 nm was determined on a spectrophotometer. The binding preference ofSiglec-10 for 2,3′-sialyllactose (2,3′PAA) and 2,6′ sialyllactose(2,6′PAA) was determined by immobilizing Siglec-10-hIg on an Immulonplate and determining the binding of the polyacrylamide biotinylatedglycoconjugates (FIG. 26). The 2,6′-PAA conjugate bound significantlygreater than either the un-sialylated lactose (negative control) or the2,3′-PAA. A subsequent cell-based experiment was done to confirm thisobservation. Full length Siglec-10 (995-2 in pcDNA3) was transfectedinto COS7 cells by DEAE-dextran method and PAA binding to transfectedcells was determined. There was significantly greater binding of the2,6′-PAA conjugate to transfected COS7 cells following sialidasepretreatment. The need for sialidase treatment suggested thatcis-binding of the Siglec-10 could inhibit interaction with the addedPAA.

EXAMPLE 16

[0336] The following example provides a description of the generation ofmonoclonal antibodies to Siglec-10 and utilization of monoclonalantibodies for detection of Siglec-10 protein by FACs analysis andWestern blotting

[0337] Balb/c mice were immunized with an intraperitoneal injection ofSiglec-10-hIg protein in Ribi Adjuvant (Corixa, Hamilton, Mont.) onceevery 3 weeks. Three days prior to sacrifice, the mice were boosted withan IV injection of Siglec-10-hIg. Splenocytes were asepticallyharvested, washed, and mixed 10:1 with mouse myeloma cells (P3x, ATCC,Rockville, Md.) in the presence of PEG 1500 50%(Roche) to induce fusion.Those clones producing antibodies selective for Siglec-10-hIg but not toother hIg, as screened by ELISA, were expanded in roller flasks. Thepurified monoclonal antibodies were further screened by Western blot ofSiglec-10-hIg and other similar fusion proteins. A third screen forantibody specificity was performed using FACs analysis of COS7 cellsthat were transfected with full length Siglec-10 expression construct.

[0338] Siglec Protein Expression.

[0339] FACs analysis of peripheral blood cell populations and cell lineswas performed to determine surface protein expression (Table 2).Anti-Siglec-10 antibody bound to isolated granulocytes (eosinophils andneutrophils) and CD 14+ monocytes with large shifts in fluorescenceintensity. The antibody did not bind to other blood cells includingCD28+ cells and CD3+ cells.

[0340] Table 2 shows expression of Siglec-10 on hematopoietic cell linesand primary leukocytes. Biotinylated monoclonal anti-Siglec-10 was addedto cells followed by treatment with FITC-conjugated streptavidin. Theantibody was chosen based on immunoreactivity to COS7 cells transfectedwith Siglec-10 as determined by FACs. For peripheral blood mononuclearcell preparations (PBMC), a secondary PE-conjugated antibody was used todistinguish sub-populations. The percentage of total cells withincreased fluorescence is indicated. Data shown represents the mean of2-3 experiments. TABLE 2 FITC Anti- Cell line Type alone (%) Siglec-10(%) Ramos B-cell (lymphoma) 0.5 89.9 THP-1 Monocyte (lymphoma) 1.7 39.1Jurkat T-cell (lymphoma) 0.4 76.6 U973 Monocyte (leukemia) 2.0 33.6 HL60Monocyte (leukemia) 0.6 93.3 K562 Monocyte (leukemia) 0.5 96.2 COS7Naive 2.4 15.4 COS7 Siglec-10 Transfected 4.7 63.3 Blood Siglec- FITC+and Population PE+ (%) FITC+ (%) PE+ (%) PBMC 19.9 CD20+  8.3 2.4 CD14+12.1 12.0 CD4lo+ 11.7 12.0 CD4hi+ 63.0 0 CD3+ 65.0 0 CD28+ 47.5 2.0Granulocytes 88.6

[0341] Western Blotting for SIGLEC-10.

[0342] Ten micrograms of cell lysates (Triton-X-100-soluble proteinfraction) from several cell lines and peripheral blood cell preparationswere mixed with sample buffer and resolved by SDS-PAGE (4-20% gradientgel) and transferred to nitrocellulose by standard western blottingtechniques. The blots were then stained with anti-Siglec-10 monoclonalantibody followed by an HRP-conjugated secondary antibody (goatanti-mouse, Biosource Int., Camarillo Calif.). Stained proteins wereimaged by adding a chemiluminescent detection reagent (Renaissance, NENBio Products, Boston, Mass.) using a Phosphorlmager 445 SI (MolecularDynamics, Sunnyvale, Calif.). The anti-Siglec-10 mAb recognized a singleband with a molecular mass of approximately 76 kDa (FIG. 27). There wereno other visible bands, implying that the antibody is specific forSiglec-10. Granulocytes and several blood cell lines appear to expressSiglec-10 (FIG. 27).

EXAMPLE 17

[0343] The following example provides a description of detection ofSiglec-10 positive hybridization signals in non-human primates (NHP) andhuman tissues using in situ hybridization.

[0344] Human ³⁵S labeled RNA probes (riboprobes) for Siglec 10 werecreated via in vitro transcription utilizing PCR product templates. Genespecific primer (GSP) sets were obtained from Life Technologies(Rockville, Md.) according to probe primer sequence data (SiglecManuscript, IIPD) for Siglec 10 L3 probe (5′ (724-744)TGCTCAGCTTCACGCCCAGAC; 3′ (1447-1456) TGCACGGAGAGGCTGAGA GA). Ampliconswere obtained from PCR amplification of full length Siglec 10 genecloned into a pSport plasmid vector. Gel electrophoresis was run andcorrect size bands were cut from gel. These bands were purified (GelSNAP Purification kit, Invitrogen, Carlsbad, Calif.) and then subclonedutilizing the TOPO-TA cloning kit (Invitrogen, Carlsbad, Calif.) intothe pCRII vector. Miniprep and sequence analyses combined with GenBankBlast were used to confirm the sequence identities (GenBank confirmed100% 274 bp identity to Chromosome 19. Separate Blast2 of novel Siglec10 sequence and miniprep sequence results gave 100% homology). Theminipreps were then PCR amplified using GSP and T7 and/or Sp6 primers.Riboprobes were produced utilizing the RNA polymerases Sp⁶ and T7 and acommercially available kit (Riboprobe® Combination System, Promega,Madison, Wis.), and in situ hybridization was performed.

[0345]FIG. 28 shows micrograph composite images of in situ hybridizationdetailing the distribution of Siglec-10 positive hybridization signalsin non-human primate and human tissues.

[0346]FIG. 28a shows Siglec-10 positive hybridization in non-humanprimate (NHP) (Panels A, C, E)/human spleen (Panels B, D, F). Panel A(Brightfield, 40× magnification) shows Lymphoid follicle (LF) andsurrounding red pulp (RP) area, NHP spleen. Panel B (Brightfield, 40×magnification) shows Lymphoid follicle (LF) and surrounding red pulparea (RP), Human spleen. Pnael C (40× magnification) is Darkfield ofPanel A showing Siglec 10 hybridization signals associated with the redpulp area (RP). Panel D (40× magnification) is Darkfield of Panel Bshowing Siglec-10 hybridization signals (white foci) in the red pulparea (arrows). Panel E (Brightfield, 200X magnification) shows detail ofred pulp showing Siglec 10 hybridization signal (black foci) associatedwith lymphocytes (arrows) and macrophages (arrowheads), NHP. Panel F(Brightfield, 200× magnification) shows detail of red pulp showingSiglec-10 hybridization signals (black foci) associated with macrophages(arrows), Human.

[0347]FIG. 28b shows Siglec-10 positive hybridization in NHP Jejunum(panels A, C, E); Human liver (B, D, F). Panel A (Brightfield, 40×magnification) shows Mucosa (M) with lymphoid follicles (LF), NHPjejunum. Panel B (Brightfield, 40× magnification) shows Liver, Human.Panel C shows darkfield of Panel A showing Siglec 10 hybridizationsignals in lymphoid follicles (arrows) and foci in lamina propria ofmucosa (arrowheads). Panel D shows darkfield of Panel B showingSiglec-10 hybridization signals along sinusoids (arrows). Panel E(Brightfield, 200× magnification) shows detail of Siglec-10hybridization signals associated with lymphocytes in lymphoid folliclesof mucosa, NHP jejunum. Panel F (Brightfield, 400× magnification) showsdetail of Siglec 10 hybridization signals (arrows) associated withKupffer cells (resident macrophages), Human liver.

[0348]FIG. 28c shows Siglec-10 positive hybridization in Non-humanPrimate Colon. Panel A (Darkfield, 40× magnification) shows transversesection of colon with mucosa (M), submucosa (SM), muscularis externa(ME) and lymphoid follicle (LF) in submucosa. Siglec 10 hybridizationsignal is present in the lamina propria of mucosa (arrows), LF ofsubmucosa (LF), and multifocally in the interstitium of muscularisextema (arrowheads). Panel B (Darkfield, 100× magnification) showsdetail of Panel A showing Siglec 10 hybridization signals in mucosallamina propria (arrows) and submucosal LF (arrowheads). Panel C(Darkfield, 100× magnification) shows detail of Panel A showing Siglec10 hybridization signals in the interstitium of the muscularis externa(arrows). Pannel D is Brightfield of Panel A showing mucosa (M),submucosa (SM) with lymphoid follicle (LF), and muscularis extema (ME).Panel D is Brightfield of Panel B showing mucosal lamina propria (LP)and lymphoid follicle (LF) in submucosa. Panel E is Brightfield of PanelC showing Siglec 10 positive mononuclear cells (arrows) in theinterstitium of the muscularis extema. Panel F (Brightfield, 200×magnification) shows detail of lamina propria of the mucosa showingSiglec 10 hybridization signals associated with lymphocytes (arrows) andmacrophages (arrowheads).

[0349]FIG. 28d shows distribution of Siglec-10 positive hybridizationsignal in NHP (panels (A, C, E)/Human Lymph Node (panels B, D, F). PanelA (Darkfield, 10× magnification) shows Siglec 10 hybridization signalsassociated with lymphoid follicles (LF). Prominent cells aremelanomacrophages (arrows), NHP lymph node. Panel B (Brightfield, 40×magnification) shows a weak Siglec 10 hybridization signals associatedwith lymphocytes, Human lymph node. Panel C is a Brightfield of Panel Ashowing LF and melanomacrophages (arrows). Panel D is a Darkfield ofPanel B showing weak Siglec 10 signal (arrow). Panel E (Brightfield,200× magnification) D depicts detail of LF showing Siglec 10hybridization signals associated with lymphocytes (arrows) andmelanomacrophages (arrowheads). Panel F (Brightfield, 400×magnification) depicts detail of LF showing weak Siglec 10 hybridizationsignals associated with lymphocytes (arrows).

[0350]FIG. 28e shows distribution of Siglec-10 positive hybridizationsignals Siglec-10 RNA Asthma Lung. Panel A (Brightfield, 100×.) showsLung parenchyma infiltrated by a mixed inflammatory cell population,which includes eosinophils, macrophages, and lymphocytes, Human lung.Panel B is Darkfield of Panel A showing multifocal Siglec 10hybridization signals (arrows). Panel C (Brightfield, 400×magnification) depicts detail of inflammatory cells of lung showingSiglec-10 hybridization signals associated with macrophages (arrows),but no signal associated with eosinophils (arrowheads).

[0351]FIG. 28f shows binding of Siglec-10 RNA to Non-human Primate(Panels A, B, D, E, G, H)/Human Lung (Panels C, F, I). Panel A(Brightfield, 40× magnification) shows Airway bronchiole (B), NHP, lung.Panel B (Brightfield, 100× magnification) shows detail of lymphoidfollicle (LF) in subbronchial area, Bronchiole (B), NHP lung. Panel C(Brightfield, 100× magnification) shows Lung parenchyma withbrown-stained alveolar macrophages (anthrosilicosis) (arrows), Humanlung. Panel D is Darkfield of Panel A showing Siglec-10 hybridizationsignals (arrows) in airway lumen (L) and lung parenchyma (arrowheads).Panel E is Darkfield of Panel B showing Siglec-10 hybridization signalin LF and in lung parenchyma (arrows). Panel F is Darkfield of Panel Cshowing Siglec-10 hybridization signals associated with alveolarmacrophages (arrows). Panel G (Brightfield, 400× magnification) depictsDetail of Panel A showing Siglec-10 hybridization signals associatedwith alveolar macrophages (arrows). Panel H (Brightfield, 400×) depictsDetail of Panel B showing Siglec 10 hybridization signals associatedwith lymphocytes of the LF (arrows) and an alveolar macrophage(arrowhead). Panel I (Brightfield, 400× magnification.) depicts Detailof Panel C showing Siglec-10 hybridization signals associated withbrown-stained (anthrosilicosis) alveolar macrophages (arrows).

[0352] The final results of in situ hybridization are described in Table3. TABLE 3 Siglec 10 ISH scores for select human and non-human primatetissues Human Non-human primate Score¹ Tissue Score Tissue Colonnormal + Lymphoid follicles (luminal aspect + + + + Lymphoid follicles,(luminal aspect of of lamina propria) lamina propria); + + TBD cellswithin the muscularis externa Colon IBD + Lymphoid follicles (luminalaspect NE² of lamina propria) Ileum + Weak lymphoid follicles, rare + +Lymphoid follicles, GALT³ lamina propria cells Jejunum − + + + Lymphoidfollicles, GALT Stomach + Weak lymphoid follicles, rare + + Lymphoidfollicles, TBD cells of lamina propria cells gastric pits Duodenum − + +Lymphoid follicle, lamina propria of mucosa/sub-mucosa + Villusenterocytes Lymph node + Rare lymphocyte + + + Lymphocyte subpopulation−? macrophage Dendritic cells/melano-macrophages Liver + + Kupffer cellsNE *Cartilage (DJD) * NE Lung - normal + + macrophages + + + Alveolarmacrophages + + Lymphoid follicle lymphocytes Airway epithelium withlymphocyte + infiltration Lung - asthma + + Inflammatory foci NE −eosinophils Spleen + + + Macrophages + + + + Red pulp very strongLymphocytes + + + Regions of lymphoid follicle

[0353] Various publications are cited herein that are herebyincorporated by reference in their entirety.

[0354] As will be apparent to those skilled in the art to which theinvention pertains, the present invention may be embodied in forms otherthan those specifically disclosed above without departing from thespirit or essential characteristics of the invention. The particularembodiments of the invention described above, are, therefore, to beconsidered as illustrative and not restrictive. The scope of the presentinvention is as set forth in the appended claims rather than beinglimited to the examples contained in the foregoing description.

1 71 1 2565 DNA Homo sapiens 1 ccacgcgtcc gggccccagg gctcagcttccgccttcggc ttccccttct gccaagagcc 60 ctgagccact cacagcacga ccagagaacaggcctgtctc aggcaggccc tgcgcctcct 120 atgcggagat gctactgcca ctgctgctgtcctcgctgct gggcgggtcc caggctatgg 180 atgggagatt ctggatacga gtgcaggagtcagtgatggt gccggagggc ctgtgcatct 240 ctgtgccctg ctctttctcc tacccccgacaagactggac agggtctacc ccagcttatg 300 gctactggtt caaagcagtg actgagacaaccaagggtgc tcctgtggcc acaaaccacc 360 agagtcgaga ggtggaaatg agcacccggggccgattcca gctcactggg gatcccgcca 420 aggggaactg ctccttggtg atcagagacgcgcagatgca ggatgagtca cagtacttct 480 ttcgggtgga gagaggaagc tatgtgagatataatttcat gaacgatggg ttctttctaa 540 aagtaacagt gctcagcttc acgcccagaccccaggacca caacaccgac ctcacctgcc 600 atgtggactt ctccagaaag ggtgtgagcgcacagaggac cgtccgactc cgtgtggcct 660 atgcccccag agaccttgtt atcagcatttcacgtgacaa cacgccagcc ctggagcccc 720 agccccaggg aaatgtccca tacctggaagcccaaaaagg ccagttcctg cggctcctct 780 gtgctgctga cagccagccc cctgccacactgagctgggt cctgcagaac agagtcctct 840 cctcgtccca tccctggggc cctagacccctggggctgga gctgcccggg gtgaaggctg 900 gggattcagg gcgctacacc tgccgagcggagaacaggct tggctcccag cagcgagccc 960 tggacctctc tgtgcagtat cctccagagaacctgagagt gatggtttcc caagcaaaca 1020 ggacagtcct ggaaaacctt gggaacggcacgtctctccc agtactggag ggccaaagcc 1080 tgtgcctggt ctgtgtcaca cacagcagccccccagccag gctgagctgg acccagaggg 1140 gacaggttct gagcccctcc cagccctcagaccccggggt cctggagctg cctcgggttc 1200 aagtggagca cgaaggagag ttcacctgccacgctcggca cccactgggc tcccagcacg 1260 tctctctcag cctctccgtg cactataagaagggactcat ctcaacggca ttctccaacg 1320 gagcgtttct gggaatcggc atcacggctcttcttttcct ctgcctggcc ctgatcatca 1380 tgaagattct accgaagaga cggactcagacagaaacccc gaggcccagg ttctcccggc 1440 acagcacgat cctggattac atcaatgtggtcccgacggc tggccccctg gctcagaagc 1500 ggaatcagaa agccacacca aacagtcctcggacccctct tccaccaggt gctccctccc 1560 cagaatcaaa gaagaaccag aaaaagcagtatcagttgcc cagtttccca gaacccaaat 1620 catccactca agccccagaa tcccaggagagccaagagga gctccattat gccacgctca 1680 acttcccagg cgtcagaccc aggcctgaggcccggatgcc caagggcacc caggcggatt 1740 atgcagaagt caagttccaa tgagggtctcttaggcttta ggactgggac ttcggctagg 1800 gaggaaggta gagtaagagg ttgaagataacagagtgcaa agtttccttc tctccctctc 1860 tctctctctt tctctctctc tctctctttctctctctttt aaaaaaacat ctggccaggg 1920 cacagtggct cacgcctgta atcccagcactttgggaggt tgaggtgggc agatcgcctg 1980 aggtcgggag ttcgagacca gcctggccaacttggtgaaa ccccatctct acaaaaaata 2040 caaaacatag ctgggcttgg tggtgtgtgcctgtagtccc agctgtcaga catttaaacc 2100 agagcaactc catctggaat aggagctgaataaaatgagg ctgagaccta ctgggctgca 2160 ttctcagaca gtggaggcat tctaagtcacaggatgagac aggaggtccg tacaagatac 2220 aggtcataaa gactttgctg ataaaacagattgcagtaaa gaagccaacc aaatcccacc 2280 aaaaccaagt tggccacgag agtgacctctggtcgtcctc actgctacac tcctgacagc 2340 accatgacag tttacaaatg ccatggcaacatcaggaagt tacccgatat gtcccaaaag 2400 ggggaggaat gaataatcca ccccttgtttagcaaataag caagaaataa ccataaaagt 2460 gggcaaccag cagctctagg cgctgctcttgtctatggag tagccattct tttgttcctt 2520 tactttctta ataaacttgc tttcaccttaaaaaaaaaaa aaaag 2565 2 2954 DNA Homo sapiens 2 tggatgggag attctggatacgagtgcagg agtcagtgat ggtgccggag ggcctgtgca 60 tctctgtgcc ctgctctttctcctaccccc gacaagactg gacagggtct accccagctt 120 atggctactg gttcaaagcagtgactgaga caaccaaggg tgctcctgtg gccacaaacc 180 accagagtcg agaggtggaaatgagcaccc ggggccgatt ccagctcact ggggatcccg 240 ccaaggggaa ctgctccttggtgatcagag acgcgcagat gcaggatgag tcacagtact 300 tctttcgggt ggagagaggaagctatgtga gatataattt catgaacgat gggttctttc 360 taaaagtaac agtgctcagcttcacgccca gaccccagga ccacaacacc gacctcacct 420 gccatgtgga cttctccagaaagggtgtga gcgcacagag gaccgtccga ctccgtgtgg 480 cctatgcccc cagagaccttgttatcagca tttcacgtga caacacgcca gccctggagc 540 cccagcccca gggaaatgtcccatacctgg aagcccaaaa aggccagttc ctgcggctcc 600 tctgtgctgc tgacagccagccccctgcca cactgagctg ggtcctgcag aacagagtcc 660 tctcctcgtc ccatccctggggccctagac ccctggggct ggagctgccc ggggtgaagg 720 ctggggattc agggcgctacacctgccgag cggagaacag gcttggctcc cagcagcgag 780 ccctggacct ctctgtgcagtatcctccag agaacctgag agtgatggtt tcccaagcaa 840 acaggacagt cctggaaaaccttgggaacg gcacgtctct cccagtactg gagggccaaa 900 gcctgtgcct ggtctgtgtcacacacagca gccccccagc caggctgagc tggacccaga 960 ggggacaggt tctgagcccctcccagccct cagaccccgg ggtcctggag ctgcctcggg 1020 ttcaagtgga gcacgaaggagagttcacct gccacgctcg gcacccactg ggctcccagc 1080 acgtctctct cagcctctccgtgcactact ccccgaagct gctgggcccc tcctgctcct 1140 gggaggctga gggtctgcactgcagctgct cctcccaggc cagcccggcc ccctctctgc 1200 gctggtggct tggggaggagctgctggagg ggaacagcag ccaggactcc ttcgaggtca 1260 cccccagctc agccgggccctgggccaaca gctccctgag cctccatgga gggctcagct 1320 ccggcctcag gctccgctgtgaggcctgga acgtccatgg ggcccagagt ggatccatcc 1380 tgcagctgcc agataagaagggactcatct caacggcatt ctccaacgga gcgtttctgg 1440 gaatcggcat cacggctcttcttttcctct gcctggccct gatcatcatg aagattctac 1500 cgaagagacg gactcagacagaaaccccga ggcccaggtt ctcccggcac agcacgatcc 1560 tggattacat caatgtggtcccgacggctg gccccctggc tcagaagcgg aatcagaaag 1620 ccacaccaaa cagtcctcggacccctcttc caccaggtgc tccctcccca gaatcaaaga 1680 agaaccagaa aaagcagtatcagttgccca gtttcccaga acccaaatca tccactcaag 1740 ccccagaatc ccaggagagccaagaggagc tccattatgc cacgctcaac ttcccaggcg 1800 tcagacccag gcctgaggcccggatgccca agggcaccca ggcggattat gcagaagtca 1860 agttccaatg agggtctcttaggctttagg actgggactt cggctaggga ggaaggtaga 1920 gtaagaggtt gaagataacagagtgcaaag tttccttctc tccctctctc tctctctttc 1980 tctctctctc tctctttctctctcttttaa aaaaacatct ggccagggca cagtggctca 2040 ctcctgtaat cccagcactttgggaggttg aggtgggcag atcgcctgag gtcgggagtt 2100 cgagaccagc ctggccaacttggtgaaacc ccgtctctac taaaaataca aaaattagct 2160 gggcatggtg gcaggcgcctgtaatcctac ctacttggga agctgaggca ggagaatcac 2220 ttgaacctgg gagacggaggttgcagtgag ccaagatcac accattgcac gccagcctgg 2280 gcaacaaagc gagactccatctcaaaaaaa aaatcctcca aatgggttgg gtgtctgtaa 2340 tcccagcact ttgggaggctaaggtgggtg gattgcttga gcccaggagt tcgagaccag 2400 cctgggcaac atggtgaaaccccatctcta caaaaaatac aaaacatagc tgggcttggt 2460 ggtgtgtgcc tgtagtcccagctgtcagac atttaaacca gagcaactcc atctggaata 2520 ggagctgaat aaaatgaggctgagacctac tgggctgcat tctcagacag tggaggcatt 2580 ctaagtcaca ggatgagacaggaggtccgt acaagataca ggtcataaag actttgctga 2640 taaaacagat tgcagtaaagaagccaacca aatcccacca aaaccaagtt ggccacgaga 2700 gtgacctctg gtcgtcctcactgctacact cctgacagca ccatgacagt ttacaaatgc 2760 catggcaaca tcaggaagttacccgatatg tcccaaaagg gggaggaatg aataatccac 2820 cccttgttta gcaaataagcaagaaataac cataaaagtg ggcaaccagc agctctaggc 2880 gctgctcttg tctatggagtagccattctt ttgttccttt actttcttaa taaacttgct 2940 ttcaccttaa aaaa 2954 32823 DNA Homo sapiens 3 ccacgcgtcc gggaagctat gtgagatata atttcatgaacgatgggttc tttctaaaag 60 taacagccct gactcagaag cctgatgtct acatccccgagaccctggag cccgggcagc 120 cggtgacggt catctgtgtg tttaactggg cctttgaggaatgtccaccc ccttctttct 180 cctggacggg ggctgccctc tcctcccaag gaaccaaaccaacgacctcc cacttctcag 240 tgctcagctt cacgcccaga ccccaggacc acaacaccgacctcacctgc catgtggact 300 tctccagaaa gggtgtgagc gtacagagga ccgtccgactccgtgtggcc tatgccccca 360 gagaccttgt tatcagcatt tcacgtgaca acacgccagccctggagccc cagccccagg 420 gaaatgtccc atacctggaa gcccaaaaag gccagttcctgcggctcctc tgtgctgctg 480 acagccagcc ccctgccaca ctgagctggg tcctgcagaacagagtcctc tcctcgtccc 540 atccctgggg ccctagaccc ctggggctgg agctgcccggggtgaaggct ggggattcag 600 ggcgctacac ctgccgagcg gagaacaggc ttggctcccagcagcgagcc ctggacctct 660 ctgtgcagta tcctccagag aacctgagag tgatggtttcccaagcaaac aggacagtcc 720 tggaaaacct tgggaacggc acgtctctcc cagtactggagggccaaagc ctgtgcctgg 780 tctgtgtcac acacagcagc cccccagcca ggctgagctggacccagagg ggacaggttc 840 tgagcccctc ccagccctca gaccccgggg tcctggagctgcctcgggtt caagtggagc 900 acgaaggaga gttcacctgc cacgctcggc acccactgggctcccagcac gtctctctca 960 gcctctccgt gcactactcc ccgaagctgc tgggcccctcctgctcctgg gaggctgagg 1020 gtctgcactg cagctgctcc tcccaggcca gcccggccccctctctgcgc tggtggcttg 1080 gggaggagct gctggagggg aacagcagcc aggactccttcgaggtcacc cccagctcag 1140 ccgggccctg ggccaacagc tccctgagcc tccatggagggctcagctcc ggcctcaggc 1200 tccgctgtga ggcctggaac gtccatgggg cccagagtggatccatcctg cagctgccag 1260 ataagaaggg actcatctca acggcattct ccaacggagcgtttctggga atcggcatca 1320 cggctcttct tttcctctgc ctggccctga tcatcatgaagattctaccg aagagacgga 1380 ctcagacaga aaccccgagg cccaggttct cccggcacagcacgatcctg gattacatca 1440 atgtggtccc gacggctggc cccctggctc agaagcggaatcagaaagcc acaccaaaca 1500 gtcctcggac ccctcttcca ccaggtgctc cctccccagaatcaaagaag aaccagaaaa 1560 agcagtatca gttgcccagt ttcccagaac ccaaatcatccactcaagcc ccagaatccc 1620 aggagagcca agaggagctc cattatgcca cgctcaacttcccaggcgtc agacccaggc 1680 ctgaggcccg gatgcccaag ggcacccagg cggattatgcagaagtcaag ttccaatgag 1740 ggtctcttag gctttaggac tgggacttcg gctagggaggaaggtagagt aagaggttga 1800 agataacaga gtgcaaagtt tccttctctc cctctctctctctctttctc tctctctctc 1860 tctttctctc tcttttaaaa aaacatctgg ccagggcacagtggctcacg cctgtaatcc 1920 cagcactttg ggaggttgag gtgggcagat cgcctgaggtcgggagttcg agaccagcct 1980 ggccaacttg gtgaaacccc gtctctacta aaaatacaaaaattagctgg gcatggtggc 2040 aggcgcctgt aatcctacct acttgggaag ctgaggcaggagaatcactt gaacctggga 2100 gacggaggtt gcagtgagcc aagatcacac cattgcatgccagcctgggc aacaaagcga 2160 gactccatct caaaaaaaaa atcctccaaa tgggttgggtgtctgtaatc ccagcacttt 2220 gggaggctaa ggtgggtgga ttgcttgagc ccaggagttcgagaccagcc tgggcaacat 2280 ggtgaaaccc catctctaca aaaaatacaa aacatagctgggcttggtgg tgtgtgcctg 2340 tagtcccagc tgtcagacat ttaaaccaga gcaactccatctggaatagg agctgaatac 2400 aatgaggctg agacctactg ggctgcattc tcagacagtggaggcattct aagtcacagg 2460 atgagacagg aggtccgtac aagatacagg tcataaagactttgctgata aaacagattg 2520 cagtaaagaa gccaaccaaa tcccaccaaa accaagttggccacgagagt gacctctggt 2580 cgtcctcact gctacactcc tgacagcacc atgacagtttacaaatgcca tggcaacatc 2640 aggaagttac ccgatatgtc ccaaaagggg gaggaatgaataatccaccc cttgtttagc 2700 aaataagcaa gaaataacca taaaagtggg caaccagcagctctaggcgc tgctcttgtc 2760 tatggagtag ccattctttt gttcctttac tttcttaataaacttgcttt caccttaaaa 2820 aaa 2823 4 1665 DNA Homo sapiens 4 cccccgacaagactggacag ggtctacccc agcttatggc tactggttca aagcagtgac 60 tgagacaaccaagggtgctc ctgtggccac aaaccaccag agtcgagagg tggaaatgag 120 cacccggggccgattccagc tcactgggga tcccgccaag gggaactgct ccttggtgat 180 cagagacgcgcagatgcagg atgagtcaca gtacttcttt cgggtggaga gaggaagcta 240 tgtgagatataatttcatga acgatgggtt ctttctaaaa gtaacagccc tgactcagaa 300 gcctgatgtctacatccccg agaccctgga gcccgggcag ccggtgacgg tcatctgtgt 360 gtttaactgggcctttgagg aatgtccacc cccttctttc tcctggacgg gggctgccct 420 ctcctcccaaggaaccaaac caacgacctc ccacttctca gtgctcagct tcacgcccag 480 accccaggaccacaacaccg acctcacctg ccatgtggac ttctccagaa agggtgtgag 540 cgtacagaggaccgtccgac tccgtgtggc ctatgccccc agagaccttg ttatcagcat 600 ttcacgtgacaacacgccag ccctggagcc ccagccccag ggaaatgtcc catacctgga 660 agcccaaaaaggccagttcc tgcggctcct ctgtgctgct gacagccagc cccctgccac 720 actgagctgggtcctgcaga acagagtcct ctcctcgtcc catccctggg gccctagacc 780 cctggggctggagctgcccg gggtgaaggc tggggattca gggcgctaca cctgccgagc 840 ggagaacaggcttggctccc agcagcgagc cctggacctc tctgtgcagt atcctccaga 900 gaacctgagagtgatggttt cccaagcaaa caggacagtc ctggaaaacc ttgggaacgg 960 cacgtctctcccagtactgg agggccaaag cctgtgcctg gtctgtgtca cacacagcag 1020 ccccccagccaggctgagct ggacccagag gggacaggtt ctgagcccct cccagccctc 1080 agaccccggggtcctggagc tgcctcgggt tcaagtggag cacgaaggag agttcacctg 1140 ccacgctcggcacccactgg gctcccagca cgtctctctc agcctctccg tgcactataa 1200 gaagggactcatctcaacgg cattctccaa cggagcgttt ctgggaatcg gcatcacggc 1260 tcttcttttcctctgcctgg ccctgatcat gtaggttaag aggaggcgtg gcggggtctg 1320 gggcctggacccaggaagag gggaggtgtt gcaggccgaa agagtgaagg tcgtgatcaa 1380 cgcagtatacctctggaggt tacatgagta aacagcaaac tgttctcata aatgcagaat 1440 gttgtccaactgacaaactg cgtctgcttc ccagagggaa tgctgagggc agtcacgccc 1500 caagcgaagtgtttcttgta attaggcaca gctgaagctt gttagtaata atatgaacct 1560 gtgatcaattaaacagctga ccaattgtta aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1620 aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaagg 1665 5 1554 DNA Homo sapiens 5ctggtgacgg tgcaggaggg cctgtgtgtc catgtgccct gctccttctc ctacccccag 60gatggctgga ctgactctga cccagttcat ggctactggt tccgggcagg agacagacca 120taccaagacg ctccagtggc cacaaacaac ccagacagag aagtgcaggc agagacccag 180ggccgattcc aactccttgg ggacatttgg agcaacgact gctccctgag catcagagac 240gccaggaaga gggataaggg gtcatatttc tttcggctag agagaggaag catgaaatgg 300agttacaaat cacagttgaa ttacaaaact aagcagctgt ctgtgtttgt gacagccctg 360acccataggc ctgacatcct catcctaggg accctagagt ctggccactc caggaacctg 420acctgctctg tgccctgggc ctgtaagcag gggacacccc ccatgatctc ctggattggg 480gcctccgtgt cctccccggg ccccactact gcccgctcct cagtgctcac ccttacccca 540aagccccagg accacggcac cagcctcacc tgtcaggtga ccttgcctgg gacaggtgtg 600accacgacca gtaccgtccg cctcgatgtg tcctaccctc cttggaactt gaccatgact 660gtcttccaag gagatgccac agcatccaca gccctgggaa atggctcatc tctttcagtc 720cttgagggcc agtctctgcg cctggtctgt gctgtcaaca gcaatccccc tgccaggctg 780agctggaccc gggggagcct gaccctgtgc ccctcacggt cctcaaaccc tgggctgctg 840gagctgcctc gagtgcacgt gagggatgaa ggggaattca cctgccgagc tcagaacgct 900cagggctccc agcacatttc cctgagcctc tccctgcaga atgagggcac aggcacctca 960agacctgtat cacaagtgac actggcagca gtcgggggag ctggagccac agccctggcc 1020ttcctgtcct tctgcatcat cttcatcata gtgaggtcct gcgggaagaa atcggcaagg 1080ccagcagcgg gcgtggggga tacaggcatg gaagatgcaa aggccatcag gggctcggcc 1140tctcagggac ccctgactga atcctggaaa gatggcaacc ccctgaagaa gcctccccca 1200gctgttgccc cctcgtcagg ggaggaagga gagctccatt atgcaaccct cagcttccat 1260aaagtgaagc ctcaggaccc gcagggacag gaggccactg acagtgaata ctcggagatc 1320aagatccaca agcgagaaac tgcagagact caggcctgtt tgaggaatca caacccctcc 1380agcaaagaag tcagaggctg attctcacag aacaagaacc ctctagagcc ccatgctatg 1440caatgtgcct ggttcccctt ccgccatgat tgtaagtttc ctgaggcctc ccccgccatg 1500tggaactgtg agttaattac acctctttca tttataaatt aaaaaaaaaa aaaa 1554 6 1676DNA Homo sapiens 6 aacagacgtt ccctcgcggc cctggcacct ctaaccccagacatgctgct gctgctgctg 60 cccctgctct gggggaggga gagggcggaa ggacagacaagtaaactgct gacgatgcag 120 agttccgtga cggtgcagga aggcctgtgt gtccatgtgccctgctcctt ctcctacccc 180 tcgcatggct ggatttaccc tggcccagta gttcatggctactggttccg ggaaggggcc 240 aatacagacc aggatgctcc agtggccaca aacaacccagctcgggcagt gtgggaggag 300 actcgggacc gattccacct ccttggggac ccacataccaagaattgcac cctgagcatc 360 agagatgcca gaagaagtga tgcggggaga tacttctttcgtatggagaa aggaagtata 420 aaatggaatt ataaacatca ccggctctct gtgaatgtgacagccttgac ccacaggccc 480 aacatcctca tcccaggcac cctggagtcc ggctgcccccagaatctgac ctgctctgtg 540 ccctgggcct gtgagcaggg gacaccccct atgatctcctggatagggac ctccgtgtcc 600 cccctggacc cctccaccac ccgctcctcg gtgctcaccctcatcccaca gccccaggac 660 catggcacca gcctcacctg tcaggtgacc ttccctggggccagcgtgac cacgaacaag 720 accgtccatc tcaacgtgtc ctacccgcct cagaacttgaccatgactgt cttccaagga 780 gacggcacag tatccacagt cttgggaaat ggctcatctctgtcactccc agagggccag 840 tctctgcgcc tggtctgtgc agttgatgca gttgacagcaatccccctgc caggctgagc 900 ctgagctgga gaggcctgac cctgtgcccc tcacagccctcaaacccggg ggtgctggag 960 ctgccttggg tgcacctgag ggatgcagct gaattcacctgcagagctca gaaccctctc 1020 ggctctcagc aggtctacct gaacgtctcc ctgcagagcaaagccacatc aggagtgact 1080 cagggggtgg tcgggggagc tggagccaca gccctggtcttcctgtcctt ctgcgtcatc 1140 ttcgttgtag tgaggtcctg caggaagaaa tcggcaaggccagcagcggg cgtgggagat 1200 acgggcatag aggatgcaaa cgctgtcagg ggttcagcctctcaggggcc cctgactgaa 1260 ccttgggcag aagacagtcc cccagaccag cctcccccagcttctgcccg ctcctcagtg 1320 ggggaaggag agctccagta tgcatccctc agcttccagatggtgaagcc ttgggactcg 1380 cggggacagg aggccactga caccgagtac tcggagatcaagatccacag atgagaaact 1440 gcagagactc accctgattg agggatcaca gcccctccaggcaagggaga agtcagaggc 1500 tgattcttgt agaattaaca gccctcaacg tgatgagctatgataacact atgaattatg 1560 tgcagagtga aaagcacaca ggctttagag tcaaagtatctcaaacctga atccacactg 1620 tgccctccct tttatttttt taactaaaag acagacaaattcctaaaaaa aaaaaa 1676 7 1831 DNA Homo sapiens 7 gaagaaccct gaggaacagacgttccctcg cggccctggc acctctaacc ccagacatgc 60 tgctgctgct gctgcccctgctctggggga gggagagggc ggaaggacag acaagtaaac 120 tgctgacgat gcagagttccgtgacggtgc aggaaggcct gtgtgtccat gtgccctgct 180 ccttctccta cccctcgcatggctggattt accctggccc agtagttcat ggctactggt 240 tccgggaagg ggccaatacagaccaggatg ctccagtggc cacaaacaac ccagctcggg 300 cagtgtggga ggagactcgggaccgattcc acctccttgg ggacccacat accgagaatt 360 gcaccctgag catcagagatgccagaagaa gtgatgcggg gagatacttc tttcgtatgg 420 agaaaggaag tataaaatggaattataaac atcaccggct ctctgtgaat gtgacagcct 480 tgacccacag gcccaacatcctcatcccag gcaccctgga gtccggctgc ccccagaatc 540 tgacctgctc tgtgccctgggcctgtgagc aggggacacc ccctatgatc tcctggatag 600 ggacctccgt gtcccccctggacccctcca ccacccgctc ctcggtgctc accctcatcc 660 cacagcccca ggaccatggcaccagcctca cctgtcaggt gaccttccct ggggccagcg 720 tgaccacgaa caagaccgtccatctcaacg tgtcctgtga gtgctgggcc gggacgcctg 780 ggtccctgat gggacccgcctcagaacttg accatgactg tcttccaagg agacggcaca 840 gtatccacag tcttgggaaatggctcatct ctgtcactcc cagagggcca gtctctgcgc 900 ctggtctgtg cagttgatgcagttgacagc aatccccctg ccaggctgag cctgagctgg 960 agaggcctga ccctgtgcccctcacagccc tcaaacccgg gggtgctgga gctgccttgg 1020 gtgcacctga gggatgaagctgaattcacc tgcagagctc agaaccctct cggctctcag 1080 caggtctacc tgaacgtctccctgcagagc aaagccacat caggagtgac tcagggggtg 1140 gtcgggggag ctggagccacagccctggtc ttcctgtcct tctgcgtcat cttcgttgta 1200 gtgaggtcct gcaggaagaaatcggcaagg ccagcagcgg gcgtgggaga tacgggcata 1260 gaggatgcaa acgctgtcaggggttcagcc tctcaggtga gtgatgtgga ctctccacag 1320 ccagcatgta gcctggacacctcccacagg atgaccccca ggactaatca gctgggcgta 1380 gccaaagtta cctcctctctgttcttcctt tcttctctgt agccccaaat cacaatgttt 1440 ggttggtttc ctcccctaagaacagctttt attgtctctg ctccctatcc tgacccttca 1500 ttgctgaggc ctgaggatctctgtcttttg ttccctcacc tgtctgcctg tctcctctcc 1560 tttcctgcct ggggggactgtccagaagac atcatcgtcc agttcctctg catttgaaca 1620 gctgttcccc cacccctcaataccgtttag agcagaagcc agcaaatact atctgtcagg 1680 gacagataga aactattttcggcttcatgg gccacacagt ctcattgcag ctcctcaaat 1740 ctgctgttgt agcaagaaagaagccatata ccctgtgtaa acaaatgaat atggctgtgt 1800 gccaataaaa ctattcacaaacataaaaaa a 1831 8 544 PRT Homo sapiens 8 Met Leu Leu Pro Leu Leu LeuSer Ser Leu Leu Gly Gly Ser Gln Ala 1 5 10 15 Met Asp Gly Arg Phe TrpIle Arg Val Gln Glu Ser Val Met Val Pro 20 25 30 Glu Gly Leu Cys Ile SerVal Pro Cys Ser Phe Ser Tyr Pro Arg Gln 35 40 45 Asp Trp Thr Gly Ser ThrPro Ala Tyr Gly Tyr Trp Phe Lys Ala Val 50 55 60 Thr Glu Thr Thr Lys GlyAla Pro Val Ala Thr Asn His Gln Ser Arg 65 70 75 80 Glu Val Glu Met SerThr Arg Gly Arg Phe Gln Leu Thr Gly Asp Pro 85 90 95 Ala Lys Gly Asn CysSer Leu Val Ile Arg Asp Ala Gln Met Gln Asp 100 105 110 Glu Ser Gln TyrPhe Phe Arg Val Glu Arg Gly Ser Tyr Val Arg Tyr 115 120 125 Asn Phe MetAsn Asp Gly Phe Phe Leu Lys Val Thr Val Leu Ser Phe 130 135 140 Thr ProArg Pro Gln Asp His Asn Thr Asp Leu Thr Cys His Val Asp 145 150 155 160Phe Ser Arg Lys Gly Val Ser Ala Gln Arg Thr Val Arg Leu Arg Val 165 170175 Ala Tyr Ala Pro Arg Asp Leu Val Ile Ser Ile Ser Arg Asp Asn Thr 180185 190 Pro Ala Leu Glu Pro Gln Pro Gln Gly Asn Val Pro Tyr Leu Glu Ala195 200 205 Gln Lys Gly Gln Phe Leu Arg Leu Leu Cys Ala Ala Asp Ser GlnPro 210 215 220 Pro Ala Thr Leu Ser Trp Val Leu Gln Asn Arg Val Leu SerSer Ser 225 230 235 240 His Pro Trp Gly Pro Arg Pro Leu Gly Leu Glu LeuPro Gly Val Lys 245 250 255 Ala Gly Asp Ser Gly Arg Tyr Thr Cys Arg AlaGlu Asn Arg Leu Gly 260 265 270 Ser Gln Gln Arg Ala Leu Asp Leu Ser ValGln Tyr Pro Pro Glu Asn 275 280 285 Leu Arg Val Met Val Ser Gln Ala AsnArg Thr Val Leu Glu Asn Leu 290 295 300 Gly Asn Gly Thr Ser Leu Pro ValLeu Glu Gly Gln Ser Leu Cys Leu 305 310 315 320 Val Cys Val Thr His SerSer Pro Pro Ala Arg Leu Ser Trp Thr Gln 325 330 335 Arg Gly Gln Val LeuSer Pro Ser Gln Pro Ser Asp Pro Gly Val Leu 340 345 350 Glu Leu Pro ArgVal Gln Val Glu His Glu Gly Glu Phe Thr Cys His 355 360 365 Ala Arg HisPro Leu Gly Ser Gln His Val Ser Leu Ser Leu Ser Val 370 375 380 His TyrLys Lys Gly Leu Ile Ser Thr Ala Phe Ser Asn Gly Ala Phe 385 390 395 400Leu Gly Ile Gly Ile Thr Ala Leu Leu Phe Leu Cys Leu Ala Leu Ile 405 410415 Ile Met Lys Ile Leu Pro Lys Arg Arg Thr Gln Thr Glu Thr Pro Arg 420425 430 Pro Arg Phe Ser Arg His Ser Thr Ile Leu Asp Tyr Ile Asn Val Val435 440 445 Pro Thr Ala Gly Pro Leu Ala Gln Lys Arg Asn Gln Lys Ala ThrPro 450 455 460 Asn Ser Pro Arg Thr Pro Leu Pro Pro Gly Ala Pro Ser ProGlu Ser 465 470 475 480 Lys Lys Asn Gln Lys Lys Gln Tyr Gln Leu Pro SerPhe Pro Glu Pro 485 490 495 Lys Ser Ser Thr Gln Ala Pro Glu Ser Gln GluSer Gln Glu Glu Leu 500 505 510 His Tyr Ala Thr Leu Asn Phe Pro Gly ValArg Pro Arg Pro Glu Ala 515 520 525 Arg Met Pro Lys Gly Thr Gln Ala AspTyr Ala Glu Val Lys Phe Gln 530 535 540 9 622 PRT Homo sapiens 9 Asp GlyArg Phe Trp Ile Arg Val Gln Glu Ser Val Met Val Pro Glu 1 5 10 15 GlyLeu Cys Ile Ser Val Pro Cys Ser Phe Ser Tyr Pro Arg Gln Asp 20 25 30 TrpThr Gly Ser Thr Pro Ala Tyr Gly Tyr Trp Phe Lys Ala Val Thr 35 40 45 GluThr Thr Lys Gly Ala Pro Val Ala Thr Asn His Gln Ser Arg Glu 50 55 60 ValGlu Met Ser Thr Arg Gly Arg Phe Gln Leu Thr Gly Asp Pro Ala 65 70 75 80Lys Gly Asn Cys Ser Leu Val Ile Arg Asp Ala Gln Met Gln Asp Glu 85 90 95Ser Gln Tyr Phe Phe Arg Val Glu Arg Gly Ser Tyr Val Arg Tyr Asn 100 105110 Phe Met Asn Asp Gly Phe Phe Leu Lys Val Thr Val Leu Ser Phe Thr 115120 125 Pro Arg Pro Gln Asp His Asn Thr Asp Leu Thr Cys His Val Asp Phe130 135 140 Ser Arg Lys Gly Val Ser Ala Gln Arg Thr Val Arg Leu Arg ValAla 145 150 155 160 Tyr Ala Pro Arg Asp Leu Val Ile Ser Ile Ser Arg AspAsn Thr Pro 165 170 175 Ala Leu Glu Pro Gln Pro Gln Gly Asn Val Pro TyrLeu Glu Ala Gln 180 185 190 Lys Gly Gln Phe Leu Arg Leu Leu Cys Ala AlaAsp Ser Gln Pro Pro 195 200 205 Ala Thr Leu Ser Trp Val Leu Gln Asn ArgVal Leu Ser Ser Ser His 210 215 220 Pro Trp Gly Pro Arg Pro Leu Gly LeuGlu Leu Pro Gly Val Lys Ala 225 230 235 240 Gly Asp Ser Gly Arg Tyr ThrCys Arg Ala Glu Asn Arg Leu Gly Ser 245 250 255 Gln Gln Arg Ala Leu AspLeu Ser Val Gln Tyr Pro Pro Glu Asn Leu 260 265 270 Arg Val Met Val SerGln Ala Asn Arg Thr Val Leu Glu Asn Leu Gly 275 280 285 Asn Gly Thr SerLeu Pro Val Leu Glu Gly Gln Ser Leu Cys Leu Val 290 295 300 Cys Val ThrHis Ser Ser Pro Pro Ala Arg Leu Ser Trp Thr Gln Arg 305 310 315 320 GlyGln Val Leu Ser Pro Ser Gln Pro Ser Asp Pro Gly Val Leu Glu 325 330 335Leu Pro Arg Val Gln Val Glu His Glu Gly Glu Phe Thr Cys His Ala 340 345350 Arg His Pro Leu Gly Ser Gln His Val Ser Leu Ser Leu Ser Val His 355360 365 Tyr Ser Pro Lys Leu Leu Gly Pro Ser Cys Ser Trp Glu Ala Glu Gly370 375 380 Leu His Cys Ser Cys Ser Ser Gln Ala Ser Pro Ala Pro Ser LeuArg 385 390 395 400 Trp Trp Leu Gly Glu Glu Leu Leu Glu Gly Asn Ser SerGln Asp Ser 405 410 415 Phe Glu Val Thr Pro Ser Ser Ala Gly Pro Trp AlaAsn Ser Ser Leu 420 425 430 Ser Leu His Gly Gly Leu Ser Ser Gly Leu ArgLeu Arg Cys Glu Ala 435 440 445 Trp Asn Val His Gly Ala Gln Ser Gly SerIle Leu Gln Leu Pro Asp 450 455 460 Lys Lys Gly Leu Ile Ser Thr Ala PheSer Asn Gly Ala Phe Leu Gly 465 470 475 480 Ile Gly Ile Thr Ala Leu LeuPhe Leu Cys Leu Ala Leu Ile Ile Met 485 490 495 Lys Ile Leu Pro Lys ArgArg Thr Gln Thr Glu Thr Pro Arg Pro Arg 500 505 510 Phe Ser Arg His SerThr Ile Leu Asp Tyr Ile Asn Val Val Pro Thr 515 520 525 Ala Gly Pro LeuAla Gln Lys Arg Asn Gln Lys Ala Thr Pro Asn Ser 530 535 540 Pro Arg ThrPro Leu Pro Pro Gly Ala Pro Ser Pro Glu Ser Lys Lys 545 550 555 560 AsnGln Lys Lys Gln Tyr Gln Leu Pro Ser Phe Pro Glu Pro Lys Ser 565 570 575Ser Thr Gln Ala Pro Glu Ser Gln Glu Ser Gln Glu Glu Leu His Tyr 580 585590 Ala Thr Leu Asn Phe Pro Gly Val Arg Pro Arg Pro Glu Ala Arg Met 595600 605 Pro Lys Gly Thr Gln Ala Asp Tyr Ala Glu Val Lys Phe Gln 610 615620 10 575 PRT Homo sapiens 10 Gly Ser Tyr Val Arg Tyr Asn Phe Met AsnAsp Gly Phe Phe Leu Lys 1 5 10 15 Val Thr Ala Leu Thr Gln Lys Pro AspVal Tyr Ile Pro Glu Thr Leu 20 25 30 Glu Pro Gly Gln Pro Val Thr Val IleCys Val Phe Asn Trp Ala Phe 35 40 45 Glu Glu Cys Pro Pro Pro Ser Phe SerTrp Thr Gly Ala Ala Leu Ser 50 55 60 Ser Gln Gly Thr Lys Pro Thr Thr SerHis Phe Ser Val Leu Ser Phe 65 70 75 80 Thr Pro Arg Pro Gln Asp His AsnThr Asp Leu Thr Cys His Val Asp 85 90 95 Phe Ser Arg Lys Gly Val Ser ValGln Arg Thr Val Arg Leu Arg Val 100 105 110 Ala Tyr Ala Pro Arg Asp LeuVal Ile Ser Ile Ser Arg Asp Asn Thr 115 120 125 Pro Ala Leu Glu Pro GlnPro Gln Gly Asn Val Pro Tyr Leu Glu Ala 130 135 140 Gln Lys Gly Gln PheLeu Arg Leu Leu Cys Ala Ala Asp Ser Gln Pro 145 150 155 160 Pro Ala ThrLeu Ser Trp Val Leu Gln Asn Arg Val Leu Ser Ser Ser 165 170 175 His ProTrp Gly Pro Arg Pro Leu Gly Leu Glu Leu Pro Gly Val Lys 180 185 190 AlaGly Asp Ser Gly Arg Tyr Thr Cys Arg Ala Glu Asn Arg Leu Gly 195 200 205Ser Gln Gln Arg Ala Leu Asp Leu Ser Val Gln Tyr Pro Pro Glu Asn 210 215220 Leu Arg Val Met Val Ser Gln Ala Asn Arg Thr Val Leu Glu Asn Leu 225230 235 240 Gly Asn Gly Thr Ser Leu Pro Val Leu Glu Gly Gln Ser Leu CysLeu 245 250 255 Val Cys Val Thr His Ser Ser Pro Pro Ala Arg Leu Ser TrpThr Gln 260 265 270 Arg Gly Gln Val Leu Ser Pro Ser Gln Pro Ser Asp ProGly Val Leu 275 280 285 Glu Leu Pro Arg Val Gln Val Glu His Glu Gly GluPhe Thr Cys His 290 295 300 Ala Arg His Pro Leu Gly Ser Gln His Val SerLeu Ser Leu Ser Val 305 310 315 320 His Tyr Ser Pro Lys Leu Leu Gly ProSer Cys Ser Trp Glu Ala Glu 325 330 335 Gly Leu His Cys Ser Cys Ser SerGln Ala Ser Pro Ala Pro Ser Leu 340 345 350 Arg Trp Trp Leu Gly Glu GluLeu Leu Glu Gly Asn Ser Ser Gln Asp 355 360 365 Ser Phe Glu Val Thr ProSer Ser Ala Gly Pro Trp Ala Asn Ser Ser 370 375 380 Leu Ser Leu His GlyGly Leu Ser Ser Gly Leu Arg Leu Arg Cys Glu 385 390 395 400 Ala Trp AsnVal His Gly Ala Gln Ser Gly Ser Ile Leu Gln Leu Pro 405 410 415 Asp LysLys Gly Leu Ile Ser Thr Ala Phe Ser Asn Gly Ala Phe Leu 420 425 430 GlyIle Gly Ile Thr Ala Leu Leu Phe Leu Cys Leu Ala Leu Ile Ile 435 440 445Met Lys Ile Leu Pro Lys Arg Arg Thr Gln Thr Glu Thr Pro Arg Pro 450 455460 Arg Phe Ser Arg His Ser Thr Ile Leu Asp Tyr Ile Asn Val Val Pro 465470 475 480 Thr Ala Gly Pro Leu Ala Gln Lys Arg Asn Gln Lys Ala Thr ProAsn 485 490 495 Ser Pro Arg Thr Pro Leu Pro Pro Gly Ala Pro Ser Pro GluSer Lys 500 505 510 Lys Asn Gln Lys Lys Gln Tyr Gln Leu Pro Ser Phe ProGlu Pro Lys 515 520 525 Ser Ser Thr Gln Ala Pro Glu Ser Gln Glu Ser GlnGlu Glu Leu His 530 535 540 Tyr Ala Thr Leu Asn Phe Pro Gly Val Arg ProArg Pro Glu Ala Arg 545 550 555 560 Met Pro Lys Gly Thr Gln Ala Asp TyrAla Glu Val Lys Phe Gln 565 570 575 11 430 PRT Homo sapiens 11 Pro ArgGln Asp Trp Thr Gly Ser Thr Pro Ala Tyr Gly Tyr Trp Phe 1 5 10 15 LysAla Val Thr Glu Thr Thr Lys Gly Ala Pro Val Ala Thr Asn His 20 25 30 GlnSer Arg Glu Val Glu Met Ser Thr Arg Gly Arg Phe Gln Leu Thr 35 40 45 GlyAsp Pro Ala Lys Gly Asn Cys Ser Leu Val Ile Arg Asp Ala Gln 50 55 60 MetGln Asp Glu Ser Gln Tyr Phe Phe Arg Val Glu Arg Gly Ser Tyr 65 70 75 80Val Arg Tyr Asn Phe Met Asn Asp Gly Phe Phe Leu Lys Val Thr Ala 85 90 95Leu Thr Gln Lys Pro Asp Val Tyr Ile Pro Glu Thr Leu Glu Pro Gly 100 105110 Gln Pro Val Thr Val Ile Cys Val Phe Asn Trp Ala Phe Glu Glu Cys 115120 125 Pro Pro Pro Ser Phe Ser Trp Thr Gly Ala Ala Leu Ser Ser Gln Gly130 135 140 Thr Lys Pro Thr Thr Ser His Phe Ser Val Leu Ser Phe Thr ProArg 145 150 155 160 Pro Gln Asp His Asn Thr Asp Leu Thr Cys His Val AspPhe Ser Arg 165 170 175 Lys Gly Val Ser Val Gln Arg Thr Val Arg Leu ArgVal Ala Tyr Ala 180 185 190 Pro Arg Asp Leu Val Ile Ser Ile Ser Arg AspAsn Thr Pro Ala Leu 195 200 205 Glu Pro Gln Pro Gln Gly Asn Val Pro TyrLeu Glu Ala Gln Lys Gly 210 215 220 Gln Phe Leu Arg Leu Leu Cys Ala AlaAsp Ser Gln Pro Pro Ala Thr 225 230 235 240 Leu Ser Trp Val Leu Gln AsnArg Val Leu Ser Ser Ser His Pro Trp 245 250 255 Gly Pro Arg Pro Leu GlyLeu Glu Leu Pro Gly Val Lys Ala Gly Asp 260 265 270 Ser Gly Arg Tyr ThrCys Arg Ala Glu Asn Arg Leu Gly Ser Gln Gln 275 280 285 Arg Ala Leu AspLeu Ser Val Gln Tyr Pro Pro Glu Asn Leu Arg Val 290 295 300 Met Val SerGln Ala Asn Arg Thr Val Leu Glu Asn Leu Gly Asn Gly 305 310 315 320 ThrSer Leu Pro Val Leu Glu Gly Gln Ser Leu Cys Leu Val Cys Val 325 330 335Thr His Ser Ser Pro Pro Ala Arg Leu Ser Trp Thr Gln Arg Gly Gln 340 345350 Val Leu Ser Pro Ser Gln Pro Ser Asp Pro Gly Val Leu Glu Leu Pro 355360 365 Arg Val Gln Val Glu His Glu Gly Glu Phe Thr Cys His Ala Arg His370 375 380 Pro Leu Gly Ser Gln His Val Ser Leu Ser Leu Ser Val His TyrLys 385 390 395 400 Lys Gly Leu Ile Ser Thr Ala Phe Ser Asn Gly Ala PheLeu Gly Ile 405 410 415 Gly Ile Thr Ala Leu Leu Phe Leu Cys Leu Ala LeuIle Met 420 425 430 12 466 PRT Homo sapiens 12 Leu Val Thr Val Gln GluGly Leu Cys Val His Val Pro Cys Ser Phe 1 5 10 15 Ser Tyr Pro Gln AspGly Trp Thr Asp Ser Asp Pro Val His Gly Tyr 20 25 30 Trp Phe Arg Ala GlyAsp Arg Pro Tyr Gln Asp Ala Pro Val Ala Thr 35 40 45 Asn Asn Pro Asp ArgGlu Val Gln Ala Glu Thr Gln Gly Arg Phe Gln 50 55 60 Leu Leu Gly Asp IleTrp Ser Asn Asp Cys Ser Leu Ser Ile Arg Asp 65 70 75 80 Ala Arg Lys ArgAsp Lys Gly Ser Tyr Phe Phe Arg Leu Glu Arg Gly 85 90 95 Ser Met Lys TrpSer Tyr Lys Ser Gln Leu Asn Tyr Lys Thr Lys Gln 100 105 110 Leu Ser ValPhe Val Thr Ala Leu Thr His Arg Pro Asp Ile Leu Ile 115 120 125 Leu GlyThr Leu Glu Ser Gly His Ser Arg Asn Leu Thr Cys Ser Val 130 135 140 ProTrp Ala Cys Lys Gln Gly Thr Pro Pro Met Ile Ser Trp Ile Gly 145 150 155160 Ala Ser Val Ser Ser Pro Gly Pro Thr Thr Ala Arg Ser Ser Val Leu 165170 175 Thr Leu Thr Pro Lys Pro Gln Asp His Gly Thr Ser Leu Thr Cys Gln180 185 190 Val Thr Leu Pro Gly Thr Gly Val Thr Thr Thr Ser Thr Val ArgLeu 195 200 205 Asp Val Ser Tyr Pro Pro Trp Asn Leu Thr Met Thr Val PheGln Gly 210 215 220 Asp Ala Thr Ala Ser Thr Ala Leu Gly Asn Gly Ser SerLeu Ser Val 225 230 235 240 Leu Glu Gly Gln Ser Leu Arg Leu Val Cys AlaVal Asn Ser Asn Pro 245 250 255 Pro Ala Arg Leu Ser Trp Thr Arg Gly SerLeu Thr Leu Cys Pro Ser 260 265 270 Arg Ser Ser Asn Pro Gly Leu Leu GluLeu Pro Arg Val His Val Arg 275 280 285 Asp Glu Gly Glu Phe Thr Cys ArgAla Gln Asn Ala Gln Gly Ser Gln 290 295 300 His Ile Ser Leu Ser Leu SerLeu Gln Asn Glu Gly Thr Gly Thr Ser 305 310 315 320 Arg Pro Val Ser GlnVal Thr Leu Ala Ala Val Gly Gly Ala Gly Ala 325 330 335 Thr Ala Leu AlaPhe Leu Ser Phe Cys Ile Ile Phe Ile Ile Val Arg 340 345 350 Ser Cys GlyLys Lys Ser Ala Arg Pro Ala Ala Gly Val Gly Asp Thr 355 360 365 Gly MetGlu Asp Ala Lys Ala Ile Arg Gly Ser Ala Ser Gln Gly Pro 370 375 380 LeuThr Glu Ser Trp Lys Asp Gly Asn Pro Leu Lys Lys Pro Pro Pro 385 390 395400 Ala Val Ala Pro Ser Ser Gly Glu Glu Gly Glu Leu His Tyr Ala Thr 405410 415 Leu Ser Phe His Lys Val Lys Pro Gln Asp Pro Gln Gly Gln Glu Ala420 425 430 Thr Asp Ser Glu Tyr Ser Glu Ile Lys Ile His Lys Arg Glu ThrAla 435 440 445 Glu Thr Gln Ala Cys Leu Arg Asn His Asn Pro Ser Ser LysGlu Val 450 455 460 Arg Gly 465 13 463 PRT Homo sapiens 13 Met Leu LeuLeu Leu Leu Pro Leu Leu Trp Gly Arg Glu Arg Ala Glu 1 5 10 15 Gly GlnThr Ser Lys Leu Leu Thr Met Gln Ser Ser Val Thr Val Gln 20 25 30 Glu GlyLeu Cys Val His Val Pro Cys Ser Phe Ser Tyr Pro Ser His 35 40 45 Gly TrpIle Tyr Pro Gly Pro Val Val His Gly Tyr Trp Phe Arg Glu 50 55 60 Gly AlaAsn Thr Asp Gln Asp Ala Pro Val Ala Thr Asn Asn Pro Ala 65 70 75 80 ArgAla Val Trp Glu Glu Thr Arg Asp Arg Phe His Leu Leu Gly Asp 85 90 95 ProHis Thr Lys Asn Cys Thr Leu Ser Ile Arg Asp Ala Arg Arg Ser 100 105 110Asp Ala Gly Arg Tyr Phe Phe Arg Met Glu Lys Gly Ser Ile Lys Trp 115 120125 Asn Tyr Lys His His Arg Leu Ser Val Asn Val Thr Ala Leu Thr His 130135 140 Arg Pro Asn Ile Leu Ile Pro Gly Thr Leu Glu Ser Gly Cys Pro Gln145 150 155 160 Asn Leu Thr Cys Ser Val Pro Trp Ala Cys Glu Gln Gly ThrPro Pro 165 170 175 Met Ile Ser Trp Ile Gly Thr Ser Val Ser Pro Leu AspPro Ser Thr 180 185 190 Thr Arg Ser Ser Val Leu Thr Leu Ile Pro Gln ProGln Asp His Gly 195 200 205 Thr Ser Leu Thr Cys Gln Val Thr Phe Pro GlyAla Ser Val Thr Thr 210 215 220 Asn Lys Thr Val His Leu Asn Val Ser TyrPro Pro Gln Asn Leu Thr 225 230 235 240 Met Thr Val Phe Gln Gly Asp GlyThr Val Ser Thr Val Leu Gly Asn 245 250 255 Gly Ser Ser Leu Ser Leu ProGlu Gly Gln Ser Leu Arg Leu Val Cys 260 265 270 Ala Val Asp Ala Val AspSer Asn Pro Pro Ala Arg Leu Ser Leu Ser 275 280 285 Trp Arg Gly Leu ThrLeu Cys Pro Ser Gln Pro Ser Asn Pro Gly Val 290 295 300 Leu Glu Leu ProTrp Val His Leu Arg Asp Ala Ala Glu Phe Thr Cys 305 310 315 320 Arg AlaGln Asn Pro Leu Gly Ser Gln Gln Val Tyr Leu Asn Val Ser 325 330 335 LeuGln Ser Lys Ala Thr Ser Gly Val Thr Gln Gly Val Val Gly Gly 340 345 350Ala Gly Ala Thr Ala Leu Val Phe Leu Ser Phe Cys Val Ile Phe Val 355 360365 Val Val Arg Ser Cys Arg Lys Lys Ser Ala Arg Pro Ala Ala Gly Val 370375 380 Gly Asp Thr Gly Ile Glu Asp Ala Asn Ala Val Arg Gly Ser Ala Ser385 390 395 400 Gln Gly Pro Leu Thr Glu Pro Trp Ala Glu Asp Ser Pro ProAsp Gln 405 410 415 Pro Pro Pro Ala Ser Ala Arg Ser Ser Val Gly Glu GlyGlu Leu Gln 420 425 430 Tyr Ala Ser Leu Ser Phe Gln Met Val Lys Pro TrpAsp Ser Arg Gly 435 440 445 Gln Glu Ala Thr Asp Thr Glu Tyr Ser Glu IleLys Ile His Arg 450 455 460 14 286 PRT Homo sapiens 14 Met Leu Leu LeuLeu Leu Pro Leu Leu Trp Gly Arg Glu Arg Ala Glu 1 5 10 15 Gly Gln ThrSer Lys Leu Leu Thr Met Gln Ser Ser Val Thr Val Gln 20 25 30 Glu Gly LeuCys Val His Val Pro Cys Ser Phe Ser Tyr Pro Ser His 35 40 45 Gly Trp IleTyr Pro Gly Pro Val Val His Gly Tyr Trp Phe Arg Glu 50 55 60 Gly Ala AsnThr Asp Gln Asp Ala Pro Val Ala Thr Asn Asn Pro Ala 65 70 75 80 Arg AlaVal Trp Glu Glu Thr Arg Asp Arg Phe His Leu Leu Gly Asp 85 90 95 Pro HisThr Glu Asn Cys Thr Leu Ser Ile Arg Asp Ala Arg Arg Ser 100 105 110 AspAla Gly Arg Tyr Phe Phe Arg Met Glu Lys Gly Ser Ile Lys Trp 115 120 125Asn Tyr Lys His His Arg Leu Ser Val Asn Val Thr Ala Leu Thr His 130 135140 Arg Pro Asn Ile Leu Ile Pro Gly Thr Leu Glu Ser Gly Cys Pro Gln 145150 155 160 Asn Leu Thr Cys Ser Val Pro Trp Ala Cys Glu Gln Gly Thr ProPro 165 170 175 Met Ile Ser Trp Ile Gly Thr Ser Val Ser Pro Leu Asp ProSer Thr 180 185 190 Thr Arg Ser Ser Val Leu Thr Leu Ile Pro Gln Pro GlnAsp His Gly 195 200 205 Thr Ser Leu Thr Cys Gln Val Thr Phe Pro Gly AlaSer Val Thr Thr 210 215 220 Asn Lys Thr Val His Leu Asn Val Ser Cys GluCys Trp Ala Gly Thr 225 230 235 240 Pro Gly Ser Leu Met Gly Pro Ala SerGlu Leu Asp His Asp Cys Leu 245 250 255 Pro Arg Arg Arg His Ser Ile HisSer Leu Gly Lys Trp Leu Ile Ser 260 265 270 Val Thr Pro Arg Gly Pro ValSer Ala Pro Gly Leu Cys Ser 275 280 285 15 2529 DNA Homo sapiens 15ccacgcgtcc gggccccagg gctcagcttc cgccttcggc ttccccttct gccaagagcc 60ctgagccact cacagcacga ccagagaaca ggcctgtctc aggcaggccc tgcgcctcct 120atgcggagat gctactgcca ctgctgctgt cctcgctgct gggcgggtcc caggctatgg 180atgggagatt ctggatacga gtgcaggagt cagtgatggt gccggagggc ctgtgcatct 240ctgtgccctg ctctttctcc tacccccgac aagactggac agggtctacc ccagcttatg 300gctactggtt caaagcagtg actgagacaa ccaagggtgc tcctgtggcc acaaaccacc 360agagtcgaga ggtggaaatg agcacccggg gccgattcca gctcactggg gatcccgcca 420aggggaactg ctccttggtg atcagagacg cgcagatgca ggatgagtca cagtacttct 480ttcgggtgga gagaggaagc tatgtgagat ataatttcat gaacgatggg ttctttctaa 540aagtaacagc cctgactcag aagcctgatg tctacatccc cgagaccctg gagcccgggc 600agccggtgac ggtcatctgt gtgtttaact gggcctttga ggaatgtcca cccccttctt 660tctcctggac gggggctgcc ctctcctccc aaggaaccaa accaacgacc tcccacttct 720cagtgctcag cttcacgccc agaccccagg accacaacac cgacctcacc tgccatgtgg 780acttctccag aaagggtgtg agcgcacaga ggaccgtccg actccgtgtg gcctatgccc 840ccagagacct tgttatcagc atttcacgtg acaacacgcc agccctggag ccccagcccc 900agggaaatgt cccatacctg gaagcccaaa aaggccagtt cctgcggctc ctctgtgctg 960ctgacagcca gccccctgcc acactgagct gggtcctgca gaacagagtc ctctcctcgt 1020cccatccctg gggccctaga cccctggggc tggagctgcc cggggtgaag gctggggatt 1080cagggcgcta cacctgccga gcggagaaca ggcttggctc ccagcagcga gccctggacc 1140tctctgtgca gtatcctcca gagaacctga gagtgatggt ttcccaagca aacaggacag 1200tcctggaaaa ccttgggaac ggcacgtctc tcccagtact ggagggccaa agcctgtgcc 1260tggtctgtgt cacacacagc agccccccag ccaggctgag ctggacccag aggggacagg 1320ttctgagccc ctcccagccc tcagaccccg gggtcctgga gctgcctcgg gttcaagtgg 1380agcacgaagg agagttcacc tgccacgctc ggcacccact gggctcccag cacgtctctc 1440tcagcctctc cgtgcactac tccccgaagc tgctgggccc ctcctgctcc tgggaggctg 1500agggtctgca ctgcagctgc tcctcccagg ccagcccggc cccctctctg cgctggtggc 1560ttggggagga gctgctggag gggaacagca gccaggactc cttcgaggtc acccccagct 1620cagccgggcc ctgggccaac agctccctga gcctccatgg agggctcagc tccggcctca 1680ggctccgctg tgaggcctgg aacgtccatg gggcccagag tggatccatc ctgcagctgc 1740cagataagaa gggactcatc tcagatccgg agcccaaatc ttgtgacaaa actcacacat 1800gcccaccgtg cccagcacct gaattcgagg gtgcaccgtc agtcttcctc ttccccccaa 1860aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg gtggtggacg 1920tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg gaggtgcata 1980atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgggtg gtcagcgtcc 2040tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag gtctccaaca 2100aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag ccccgagaac 2160cacaggtgta caccctgccc ccatcccggg atgagctgac caagaaccag gtcagcctga 2220cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag agcaatgggc 2280agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc tccttcttcc 2340tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc ttctcatgct 2400ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc ctgtctccgg 2460gtaaatgagt gcgacggccg gcaagccccg ctccccgggc tctcgcggtc gcacgaggat 2520gcttctaga 2529 16 779 PRT Artificial Sequence Description of ArtificialSequence L3-hIg 16 Met Leu Leu Pro Leu Leu Leu Ser Ser Leu Leu Gly GlySer Gln Ala 1 5 10 15 Met Asp Gly Arg Phe Trp Ile Arg Val Gln Glu SerVal Met Val Pro 20 25 30 Glu Gly Leu Cys Ile Ser Val Pro Cys Ser Phe SerTyr Pro Arg Gln 35 40 45 Asp Trp Thr Gly Ser Thr Pro Ala Tyr Gly Tyr TrpPhe Lys Ala Val 50 55 60 Thr Glu Thr Thr Lys Gly Ala Pro Val Ala Thr AsnHis Gln Ser Arg 65 70 75 80 Glu Val Glu Met Ser Thr Arg Gly Arg Phe GlnLeu Thr Gly Asp Pro 85 90 95 Ala Lys Gly Asn Cys Ser Leu Val Ile Arg AspAla Gln Met Gln Asp 100 105 110 Glu Ser Gln Tyr Phe Phe Arg Val Glu ArgGly Ser Tyr Val Arg Tyr 115 120 125 Asn Phe Met Asn Asp Gly Phe Phe LeuLys Val Thr Ala Leu Thr Gln 130 135 140 Lys Pro Asp Val Tyr Ile Pro GluThr Leu Glu Pro Gly Gln Pro Val 145 150 155 160 Thr Val Ile Cys Val PheAsn Trp Ala Phe Glu Glu Cys Pro Pro Pro 165 170 175 Ser Phe Ser Trp ThrGly Ala Ala Leu Ser Ser Gln Gly Thr Lys Pro 180 185 190 Thr Thr Ser HisPhe Ser Val Leu Ser Phe Thr Pro Arg Pro Gln Asp 195 200 205 His Asn ThrAsp Leu Thr Cys His Val Asp Phe Ser Arg Lys Gly Val 210 215 220 Ser AlaGln Arg Thr Val Arg Leu Arg Val Ala Tyr Ala Pro Arg Asp 225 230 235 240Leu Val Ile Ser Ile Ser Arg Asp Asn Thr Pro Ala Leu Glu Pro Gln 245 250255 Pro Gln Gly Asn Val Pro Tyr Leu Glu Ala Gln Lys Gly Gln Phe Leu 260265 270 Arg Leu Leu Cys Ala Ala Asp Ser Gln Pro Pro Ala Thr Leu Ser Trp275 280 285 Val Leu Gln Asn Arg Val Leu Ser Ser Ser His Pro Trp Gly ProArg 290 295 300 Pro Leu Gly Leu Glu Leu Pro Gly Val Lys Ala Gly Asp SerGly Arg 305 310 315 320 Tyr Thr Cys Arg Ala Glu Asn Arg Leu Gly Ser GlnGln Arg Ala Leu 325 330 335 Asp Leu Ser Val Gln Tyr Pro Pro Glu Asn LeuArg Val Met Val Ser 340 345 350 Gln Ala Asn Arg Thr Val Leu Glu Asn LeuGly Asn Gly Thr Ser Leu 355 360 365 Pro Val Leu Glu Gly Gln Ser Leu CysLeu Val Cys Val Thr His Ser 370 375 380 Ser Pro Pro Ala Arg Leu Ser TrpThr Gln Arg Gly Gln Val Leu Ser 385 390 395 400 Pro Ser Gln Pro Ser AspPro Gly Val Leu Glu Leu Pro Arg Val Gln 405 410 415 Val Glu His Glu GlyGlu Phe Thr Cys His Ala Arg His Pro Leu Gly 420 425 430 Ser Gln His ValSer Leu Ser Leu Ser Val His Tyr Ser Pro Lys Leu 435 440 445 Leu Gly ProSer Cys Ser Trp Glu Ala Glu Gly Leu His Cys Ser Cys 450 455 460 Ser SerGln Ala Ser Pro Ala Pro Ser Leu Arg Trp Trp Leu Gly Glu 465 470 475 480Glu Leu Leu Glu Gly Asn Ser Ser Gln Asp Ser Phe Glu Val Thr Pro 485 490495 Ser Ser Ala Gly Pro Trp Ala Asn Ser Ser Leu Ser Leu His Gly Gly 500505 510 Leu Ser Ser Gly Leu Arg Leu Arg Cys Glu Ala Trp Asn Val His Gly515 520 525 Ala Gln Ser Gly Ser Ile Leu Gln Leu Pro Asp Lys Lys Gly LeuIle 530 535 540 Ser Asp Pro Glu Pro Lys Ser Cys Asp Lys Thr His Thr CysPro Pro 545 550 555 560 Cys Pro Ala Pro Glu Phe Glu Gly Ala Pro Ser ValPhe Leu Phe Pro 565 570 575 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser ArgThr Pro Glu Val Thr 580 585 590 Cys Val Val Val Asp Val Ser His Glu AspPro Glu Val Lys Phe Asn 595 600 605 Trp Tyr Val Asp Gly Val Glu Val HisAsn Ala Lys Thr Lys Pro Arg 610 615 620 Glu Glu Gln Tyr Asn Ser Thr TyrArg Val Val Ser Val Leu Thr Val 625 630 635 640 Leu His Gln Asp Trp LeuAsn Gly Lys Glu Tyr Lys Cys Lys Val Ser 645 650 655 Asn Lys Ala Leu ProAla Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 660 665 670 Gly Gln Pro ArgGlu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 675 680 685 Glu Leu ThrLys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 690 695 700 Tyr ProSer Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 705 710 715 720Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 725 730735 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 740745 750 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr755 760 765 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 770 775 17 1053DNA Artificial Sequence Description of Artificial Sequence L3cyto-wt 17atgtccccta tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt 60ttggaatatc ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa 120tggcgaaaca aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat 180ggtgatgtta aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac 240atgttgggtg gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg 300gatattagat acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt 360gattttctta gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa 420acatatttaa atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat 480gttgttttat acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa 540aaacgtattg aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca 600tggcctttgc agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat 660ctggttccgc gtggatcccc gaattccaag agacggactc agacagaaac cccgaggccc 720aggttctccc ggcacagcac gatcctggat tacatcaatg tggtcccgac ggctggcccc 780ctggctcaga agcggaatca gaaagccaca ccaaacagtc ctcggacccc tcttccacca 840ggtgctccct ccccagaatc aaagaagaac cagaaaaagc agtatcagtt gcccagtttc 900ccagaaccca aatcatccac tcaagcccca gaatcccagg agagccaaga ggagctccat 960tatgccacgc tcaacttccc aggcgtcaga cccaggcctg aggcccggat gcccaagggc 1020acccaggcgg attatgcaga agtcaagttc caa 1053 18 1053 DNA ArtificialSequence Description of Artificial Sequence L3cyto- Y641F 18 atgtcccctatactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt 60 ttggaatatcttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa 120 tggcgaaacaaaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat 180 ggtgatgttaaattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac 240 atgttgggtggttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg 300 gatattagatacggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt 360 gattttcttagcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa 420 acatatttaaatggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat 480 gttgttttatacatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa 540 aaacgtattgaagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca 600 tggcctttgcagggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat 660 ctggttccgcgtggatcccc gaattccaag agacggactc agacagaaac cccgaggccc 720 aggttctcccggcacagcac gatcctggat tacatcaatg tggtcccgac ggctggcccc 780 ctggctcagaagcggaatca gaaagccaca ccaaacagtc ctcggacccc tcttccacca 840 ggtgctccctccccagaatc aaagaagaac cagaaaaagc agtttcagtt gcccagtttc 900 ccagaacccaaatcatccac tcaagcccca gaatcccagg agagccaaga ggagctccat 960 tatgccacgctcaacttccc aggcgtcaga cccaggcctg aggcccggat gcccaagggc 1020 acccaggcggattatgcaga agtcaagttc caa 1053 19 1053 DNA Artificial SequenceDescription of Artificial Sequence L3cyto- Y667F 19 atgtcccctatactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt 60 ttggaatatcttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa 120 tggcgaaacaaaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat 180 ggtgatgttaaattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac 240 atgttgggtggttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg 300 gatattagatacggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt 360 gattttcttagcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa 420 acatatttaaatggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat 480 gttgttttatacatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa 540 aaacgtattgaagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca 600 tggcctttgcagggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat 660 ctggttccgcgtggatcccc gaattccaag agacggactc agacagaaac cccgaggccc 720 aggttctcccggcacagcac gatcctggat tacatcaatg tggtcccgac ggctggcccc 780 ctggctcagaagcggaatca gaaagccaca ccaaacagtc ctcggacccc tcttccacca 840 ggtgctccctccccagaatc aaagaagaac cagaaaaagc agtatcagtt gcccagtttc 900 ccagaacccaaatcatccac tcaagcccca gaatcccagg agagccaaga ggagctccat 960 tttgccacgctcaacttccc aggcgtcaga cccaggcctg aggcccggat gcccaagggc 1020 acccaggcggattatgcaga agtcaagttc caa 1053 20 750 DNA Artificial SequenceDescription of Artificial Sequence L3cyto- Y691F 20 atgtcccctatactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt 60 ttggaatatcttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa 120 tggcgaaacaaaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat 180 ggtgatgttaaattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac 240 atgttgggtggttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg 300 gatattagatacggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt 360 gattttcttagcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa 420 acatatttaaatggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat 480 gttgttttatacatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa 540 aaacgtattgaagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca 600 tggcctttgcagggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat 660 ctggttccgcgtggatcccc gaattccaag agacggactc agacagaaac cccgaggccc 720 aggttctcccggcacagcac gatcctggat 750 21 894 DNA Artificial Sequence Description ofArtificial Sequence L3cyto-Y641 alone 21 atgtccccta tactaggttattggaaaatt aagggccttg tgcaacccac tcgacttctt 60 ttggaatatc ttgaagaaaaatatgaagag catttgtatg agcgcgatga aggtgataaa 120 tggcgaaaca aaaagtttgaattgggtttg gagtttccca atcttcctta ttatattgat 180 ggtgatgtta aattaacacagtctatggcc atcatacgtt atatagctga caagcacaac 240 atgttgggtg gttgtccaaaagagcgtgca gagatttcaa tgcttgaagg agcggttttg 300 gatattagat acggtgtttcgagaattgca tatagtaaag actttgaaac tctcaaagtt 360 gattttctta gcaagctacctgaaatgctg aaaatgttcg aagatcgttt atgtcataaa 420 acatatttaa atggtgatcatgtaacccat cctgacttca tgttgtatga cgctcttgat 480 gttgttttat acatggacccaatgtgcctg gatgcgttcc caaaattagt ttgttttaaa 540 aaacgtattg aagctatcccacaaattgat aagtacttga aatccagcaa gtatatagca 600 tggcctttgc agggctggcaagccacgttt ggtggtggcg accatcctcc aaaatcggat 660 ctggttccgc gtggatccccgaattccatc aatgtggtcc cgacggctgg ccccctggct 720 cagaagcgga atcagaaagccacaccaaac agtcctcgga cccctcttcc accaggtgct 780 ccctccccag aatcaaagaagaaccagaaa aagcagtatc agttgcccag tttcccagaa 840 cccaaatcat ccactcaagccccagaatcc caggagagcc aagaggagct ccat 894 22 348 PRT Artificial SequenceDescription of Artificial Sequence L3cyto-wt 22 Met Ser Pro Ile Leu GlyTyr Trp Lys Ile Lys Gly Leu Val Gln Pro 1 5 10 15 Thr Arg Leu Leu LeuGlu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu 20 25 30 Tyr Glu Arg Asp GluGly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu 35 40 45 Gly Leu Glu Phe ProAsn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys 50 55 60 Leu Thr Gln Ser MetAla Ile Ile Arg Tyr Ile Ala Asp Lys His Asn 65 70 75 80 Met Leu Gly GlyCys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu 85 90 95 Gly Ala Val LeuAsp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser 100 105 110 Lys Asp PheGlu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu 115 120 125 Met LeuLys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn 130 135 140 GlyAsp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp 145 150 155160 Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu 165170 175 Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr180 185 190 Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp GlnAla 195 200 205 Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu ValPro Arg 210 215 220 Gly Ser Pro Asn Ser Lys Arg Arg Thr Gln Thr Glu ThrPro Arg Pro 225 230 235 240 Arg Phe Ser Arg His Ser Thr Ile Leu Asp TyrIle Asn Val Val Pro 245 250 255 Thr Ala Gly Pro Leu Ala Gln Lys Lys AlaThr Pro Asn Ser Pro Arg 260 265 270 Thr Pro Leu Pro Pro Gly Ala Pro SerPro Glu Ser Lys Lys Asn Gln 275 280 285 Lys Lys Gln Tyr Gln Leu Pro SerPhe Pro Glu Pro Lys Ser Ser Thr 290 295 300 Gln Ala Pro Glu Ser Gln GluSer Gln Glu Glu Leu His Tyr Ala Thr 305 310 315 320 Leu Asn Phe Pro GlyVal Arg Pro Arg Pro Glu Ala Arg Met Pro Lys 325 330 335 Gly Thr Gln AlaAsp Tyr Ala Glu Val Lys Phe Gln 340 345 23 348 PRT Artificial SequenceDescription of Artificial Sequence L3cyto- Y641F 23 Met Ser Pro Ile LeuGly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro 1 5 10 15 Thr Arg Leu LeuLeu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu 20 25 30 Tyr Glu Arg AspGlu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu 35 40 45 Gly Leu Glu PhePro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys 50 55 60 Leu Thr Gln SerMet Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn 65 70 75 80 Met Leu GlyGly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu 85 90 95 Gly Ala ValLeu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser 100 105 110 Lys AspPhe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu 115 120 125 MetLeu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn 130 135 140Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp 145 150155 160 Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu165 170 175 Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp LysTyr 180 185 190 Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly TrpGln Ala 195 200 205 Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp LeuVal Pro Arg 210 215 220 Gly Ser Pro Asn Ser Lys Arg Arg Thr Gln Thr GluThr Pro Arg Pro 225 230 235 240 Arg Phe Ser Arg His Ser Thr Ile Leu AspTyr Ile Asn Val Val Pro 245 250 255 Thr Ala Gly Pro Leu Ala Gln Lys LysAla Thr Pro Asn Ser Pro Arg 260 265 270 Thr Pro Leu Pro Pro Gly Ala ProSer Pro Glu Ser Lys Lys Asn Gln 275 280 285 Lys Lys Gln Phe Gln Leu ProSer Phe Pro Glu Pro Lys Ser Ser Thr 290 295 300 Gln Ala Pro Glu Ser GlnGlu Ser Gln Glu Glu Leu His Tyr Ala Thr 305 310 315 320 Leu Asn Phe ProGly Val Arg Pro Arg Pro Glu Ala Arg Met Pro Lys 325 330 335 Gly Thr GlnAla Asp Tyr Ala Glu Val Lys Phe Gln 340 345 24 348 PRT ArtificialSequence Description of Artificial Sequence L3cyto- Y667F 24 Met Ser ProIle Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro 1 5 10 15 Thr ArgLeu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu 20 25 30 Tyr GluArg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu 35 40 45 Gly LeuGlu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys 50 55 60 Leu ThrGln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn 65 70 75 80 MetLeu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu 85 90 95 GlyAla Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser 100 105 110Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu 115 120125 Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn 130135 140 Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp145 150 155 160 Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe ProLys Leu 165 170 175 Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln IleAsp Lys Tyr 180 185 190 Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu GlnGly Trp Gln Ala 195 200 205 Thr Phe Gly Gly Gly Asp His Pro Pro Lys SerAsp Leu Val Pro Arg 210 215 220 Gly Ser Pro Asn Ser Lys Arg Arg Thr GlnThr Glu Thr Pro Arg Pro 225 230 235 240 Arg Phe Ser Arg His Ser Thr IleLeu Asp Tyr Ile Asn Val Val Pro 245 250 255 Thr Ala Gly Pro Leu Ala GlnLys Lys Ala Thr Pro Asn Ser Pro Arg 260 265 270 Thr Pro Leu Pro Pro GlyAla Pro Ser Pro Glu Ser Lys Lys Asn Gln 275 280 285 Lys Lys Gln Tyr GlnLeu Pro Ser Phe Pro Glu Pro Lys Ser Ser Thr 290 295 300 Gln Ala Pro GluSer Gln Glu Ser Gln Glu Glu Leu His Phe Ala Thr 305 310 315 320 Leu AsnPhe Pro Gly Val Arg Pro Arg Pro Glu Ala Arg Met Pro Lys 325 330 335 GlyThr Gln Ala Asp Tyr Ala Glu Val Lys Phe Gln 340 345 25 348 PRTArtificial Sequence Description of Artificial Sequence L3cyto- Y691F 25Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro 1 5 1015 Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu 20 2530 Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu 35 4045 Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys 50 5560 Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn 65 7075 80 Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu 8590 95 Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser100 105 110 Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu ProGlu 115 120 125 Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr TyrLeu Asn 130 135 140 Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr AspAla Leu Asp 145 150 155 160 Val Val Leu Tyr Met Asp Pro Met Cys Leu AspAla Phe Pro Lys Leu 165 170 175 Val Cys Phe Lys Lys Arg Ile Glu Ala IlePro Gln Ile Asp Lys Tyr 180 185 190 Leu Lys Ser Ser Lys Tyr Ile Ala TrpPro Leu Gln Gly Trp Gln Ala 195 200 205 Thr Phe Gly Gly Gly Asp His ProPro Lys Ser Asp Leu Val Pro Arg 210 215 220 Gly Ser Pro Asn Ser Lys ArgArg Thr Gln Thr Glu Thr Pro Arg Pro 225 230 235 240 Arg Phe Ser Arg HisSer Thr Ile Leu Asp Tyr Ile Asn Val Val Pro 245 250 255 Thr Ala Gly ProLeu Ala Gln Lys Lys Ala Thr Pro Asn Ser Pro Arg 260 265 270 Thr Pro LeuPro Pro Gly Ala Pro Ser Pro Glu Ser Lys Lys Asn Gln 275 280 285 Lys LysGln Tyr Gln Leu Pro Ser Phe Pro Glu Pro Lys Ser Ser Thr 290 295 300 GlnAla Pro Glu Ser Gln Glu Ser Gln Glu Glu Leu His Tyr Ala Thr 305 310 315320 Leu Asn Phe Pro Gly Val Arg Pro Arg Pro Glu Ala Arg Met Pro Lys 325330 335 Gly Thr Gln Ala Asp Phe Ala Glu Val Lys Phe Gln 340 345 26 298PRT Artificial Sequence Description of Artificial Sequence L3cyto-Y641alone 26 Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro1 5 10 15 Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu HisLeu 20 25 30 Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe GluLeu 35 40 45 Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp ValLys 50 55 60 Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys HisAsn 65 70 75 80 Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser MetLeu Glu 85 90 95 Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile AlaTyr Ser 100 105 110 Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser LysLeu Pro Glu 115 120 125 Met Leu Lys Met Phe Glu Asp Arg Leu Cys His LysThr Tyr Leu Asn 130 135 140 Gly Asp His Val Thr His Pro Asp Phe Met LeuTyr Asp Ala Leu Asp 145 150 155 160 Val Val Leu Tyr Met Asp Pro Met CysLeu Asp Ala Phe Pro Lys Leu 165 170 175 Val Cys Phe Lys Lys Arg Ile GluAla Ile Pro Gln Ile Asp Lys Tyr 180 185 190 Leu Lys Ser Ser Lys Tyr IleAla Trp Pro Leu Gln Gly Trp Gln Ala 195 200 205 Thr Phe Gly Gly Gly AspHis Pro Pro Lys Ser Asp Leu Val Pro Arg 210 215 220 Gly Ser Pro Asn SerIle Asn Val Val Pro Thr Ala Gly Pro Leu Ala 225 230 235 240 Gln Lys ArgAsn Gln Lys Ala Thr Pro Asn Ser Pro Arg Thr Pro Leu 245 250 255 Pro ProGly Ala Pro Ser Pro Glu Ser Lys Lys Asn Gln Lys Lys Gln 260 265 270 TyrGln Leu Pro Ser Phe Pro Glu Pro Lys Ser Ser Thr Gln Ala Pro 275 280 285Glu Ser Gln Glu Ser Gln Glu Glu Leu His 290 295 27 3024 DNA ArtificialSequence Description of Artificial Sequence Siglec-BMSL3-995-2 27ccacgcgtcc gggccccagg gctcagcttc cgccttcggc ttccccttct gccaagagcc 60ctgagccact cacagcacga ccagagaaca ggcctgtctc aggcaggccc tgcgcctcct 120atgcggagat gctactgcca ctgctgctgt cctcgctgct gggcgggtcc caggctatgg 180atgggagatt ctggatacga gtgcaggagt cagtgatggt gccggagggc ctgtgcatct 240ctgtgccctg ctctttctcc tacccccgac aagactggac agggtctacc ccagcttatg 300gctactggtt caaagcagtg actgagacaa ccaagggtgc tcctgtggcc acaaaccacc 360agagtcgaga ggtggaaatg agcacccggg gccgattcca gctcactggg gatcccgcca 420aggggaactg ctccttggtg atcagagacg cgcagatgca ggatgagtca cagtacttct 480ttcgggtgga gagaggaagc tatgtgagat ataatttcat gaacgatggg ttctttctaa 540aagtaacagc cctgactcag aagcctgatg tctacatccc cgagaccctg gagcccgggc 600agccggtgac ggtcatctgt gtgtttaact gggcctttga ggaatgtcca cccccttctt 660tctcctggac gggggctgcc ctctcctccc aaggaaccaa accaacgacc tcccacttct 720cagtgctcag cttcacgccc agaccccagg accacaacac cgacctcacc tgccatgtgg 780acttctccag aaagggtgtg agcgcacaga ggaccgtccg actccgtgtg gcctatgccc 840ccagagacct tgttatcagc atttcacgtg acaacacgcc agccctggag ccccagcccc 900agggaaatgt cccatacctg gaagcccaaa aaggccagtt cctgcggctc ctctgtgctg 960ctgacagcca gccccctgcc acactgagct gggtcctgca gaacagagtc ctctcctcgt 1020cccatccctg gggccctaga cccctggggc tggagctgcc cggggtgaag gctggggatt 1080cagggcgcta cacctgccga gcggagaaca ggcttggctc ccagcagcga gccctggacc 1140tctctgtgca gtatcctcca gagaacctga gagtgatggt ttcccaagca aacaggacag 1200tcctggaaaa ccttgggaac ggcacgtctc tcccagtact ggagggccaa agcctgtgcc 1260tggtctgtgt cacacacagc agccccccag ccaggctgag ctggacccag aggggacagg 1320ttctgagccc ctcccagccc tcagaccccg gggtcctgga gctgcctcgg gttcaagtgg 1380agcacgaagg agagttcacc tgccacgctc ggcacccact gggctcccag cacgtctctc 1440tcagcctctc cgtgcactac tccccgaagc tgctgggccc ctcctgctcc tgggaggctg 1500agggtctgca ctgcagctgc tcctcccagg ccagcccggc cccctctctg cgctggtggc 1560ttggggagga gctgctggag gggaacagca gccaggactc cttcgaggtc acccccagct 1620cagccgggcc ctgggccaac agctccctga gcctccatgg agggctcagc tccggcctca 1680ggctccgctg tgaggcctgg aacgtccatg gggcccagag tggatccatc ctgcagctgc 1740cagataagaa gggactcatc tcaacggcat tctccaacgg agcgtttctg ggaatcggca 1800tcacggctct tcttttcctc tgcctggccc tgatcatcat gaagattcta ccgaagagac 1860ggactcagac agaaaccccg aggcccaggt tctcccggca cagcacgatc ctggattaca 1920tcaatgtggt cccgacggct ggccccctgg ctcagaagcg gaatcagaaa gccacaccaa 1980acagtcctcg gacccctctt ccaccaggtg ctccctcccc agaatcaaag aagaaccaga 2040aaaagcagta tcagttgccc agtttcccag aacccaaatc atccactcaa gccccagaat 2100cccaggagag ccaagaggag ctccattatg ccacgctcaa cttcccaggc gtcagaccca 2160ggcctgaggc ccggatgccc aagggcaccc aggcggatta tgcagaagtc aagttccaat 2220gagggtctct taggctttag gactgggact tcggctaggg aggaaggtag agtaagaggt 2280tgaagataac agagtgcaaa gtttccttct ctccctctct ctctctcttt ctctctctct 2340ctctctttct ctctctttta aaaaaacatc tggccagggc acagtggctc acgcctgtaa 2400tcccagcact ttgggaggtt gaggtgggca gatcgcctga ggtcgggagt tcgagaccag 2460cctggccaac ttggtgaaac cccatctcta caaaaaatac aaaacatagc tgggcttggt 2520ggtgtgtgcc tgtagtccca gctgtcagac atttaaacca gagcaactcc atctggaata 2580ggagctgaat aaaatgaggc tgagacctac tgggctgcat tctcagacag tggaggcatt 2640ctaagtcaca ggatgagaca ggaggtccgt acaagataca ggtcataaag actttgctga 2700taaaacagat tgcagtaaag aagccaacca aatcccacca aaaccaagtt ggccacgaga 2760gtgacctctg gtcgtcctca ctgctacact cctgacagca ccatgacagt ttacaaatgc 2820catggcaaca tcaggaagtt acccgatatg tcccaaaagg gggaggaatg aataatccac 2880cccttgttta gcaaataagc aagaaataac cataaaagtg ggcaaccagc agctctaggc 2940gctgctcttg tctatggagt agccattctt ttgttccttt actttcttaa taaacttgct 3000ttcaccttaa aaaaaaaaaa aaag 3024 28 697 PRT Artificial SequenceDescription of Artificial Sequence Siglec-BMSL3-995-2 28 Met Leu Leu ProLeu Leu Leu Ser Ser Leu Leu Gly Gly Ser Gln Ala 1 5 10 15 Met Asp GlyArg Phe Trp Ile Arg Val Gln Glu Ser Val Met Val Pro 20 25 30 Glu Gly LeuCys Ile Ser Val Pro Cys Ser Phe Ser Tyr Pro Arg Gln 35 40 45 Asp Trp ThrGly Ser Thr Pro Ala Tyr Gly Tyr Trp Phe Lys Ala Val 50 55 60 Thr Glu ThrThr Lys Gly Ala Pro Val Ala Thr Asn His Gln Ser Arg 65 70 75 80 Glu ValGlu Met Ser Thr Arg Gly Arg Phe Gln Leu Thr Gly Asp Pro 85 90 95 Ala LysGly Asn Cys Ser Leu Val Ile Arg Asp Ala Gln Met Gln Asp 100 105 110 GluSer Gln Tyr Phe Phe Arg Val Glu Arg Gly Ser Tyr Val Arg Tyr 115 120 125Asn Phe Met Asn Asp Gly Phe Phe Leu Lys Val Thr Ala Leu Thr Gln 130 135140 Lys Pro Asp Val Tyr Ile Pro Glu Thr Leu Glu Pro Gly Gln Pro Val 145150 155 160 Thr Val Ile Cys Val Phe Asn Trp Ala Phe Glu Glu Cys Pro ProPro 165 170 175 Ser Phe Ser Trp Thr Gly Ala Ala Leu Ser Ser Gln Gly ThrLys Pro 180 185 190 Thr Thr Ser His Phe Ser Val Leu Ser Phe Thr Pro ArgPro Gln Asp 195 200 205 His Asn Thr Asp Leu Thr Cys His Val Asp Phe SerArg Lys Gly Val 210 215 220 Ser Ala Gln Arg Thr Val Arg Leu Arg Val AlaTyr Ala Pro Arg Asp 225 230 235 240 Leu Val Ile Ser Ile Ser Arg Asp AsnThr Pro Ala Leu Glu Pro Gln 245 250 255 Pro Gln Gly Asn Val Pro Tyr LeuGlu Ala Gln Lys Gly Gln Phe Leu 260 265 270 Arg Leu Leu Cys Ala Ala AspSer Gln Pro Pro Ala Thr Leu Ser Trp 275 280 285 Val Leu Gln Asn Arg ValLeu Ser Ser Ser His Pro Trp Gly Pro Arg 290 295 300 Pro Leu Gly Leu GluLeu Pro Gly Val Lys Ala Gly Asp Ser Gly Arg 305 310 315 320 Tyr Thr CysArg Ala Glu Asn Arg Leu Gly Ser Gln Gln Arg Ala Leu 325 330 335 Asp LeuSer Val Gln Tyr Pro Pro Glu Asn Leu Arg Val Met Val Ser 340 345 350 GlnAla Asn Arg Thr Val Leu Glu Asn Leu Gly Asn Gly Thr Ser Leu 355 360 365Pro Val Leu Glu Gly Gln Ser Leu Cys Leu Val Cys Val Thr His Ser 370 375380 Ser Pro Pro Ala Arg Leu Ser Trp Thr Gln Arg Gly Gln Val Leu Ser 385390 395 400 Pro Ser Gln Pro Ser Asp Pro Gly Val Leu Glu Leu Pro Arg ValGln 405 410 415 Val Glu His Glu Gly Glu Phe Thr Cys His Ala Arg His ProLeu Gly 420 425 430 Ser Gln His Val Ser Leu Ser Leu Ser Val His Tyr SerPro Lys Leu 435 440 445 Leu Gly Pro Ser Cys Ser Trp Glu Ala Glu Gly LeuHis Cys Ser Cys 450 455 460 Ser Ser Gln Ala Ser Pro Ala Pro Ser Leu ArgTrp Trp Leu Gly Glu 465 470 475 480 Glu Leu Leu Glu Gly Asn Ser Ser GlnAsp Ser Phe Glu Val Thr Pro 485 490 495 Ser Ser Ala Gly Pro Trp Ala AsnSer Ser Leu Ser Leu His Gly Gly 500 505 510 Leu Ser Ser Gly Leu Arg LeuArg Cys Glu Ala Trp Asn Val His Gly 515 520 525 Ala Gln Ser Gly Ser IleLeu Gln Leu Pro Asp Lys Lys Gly Leu Ile 530 535 540 Ser Thr Ala Phe SerAsn Gly Ala Phe Leu Gly Ile Gly Ile Thr Ala 545 550 555 560 Leu Leu PheLeu Cys Leu Ala Leu Ile Ile Met Lys Ile Leu Pro Lys 565 570 575 Arg ArgThr Gln Thr Glu Thr Pro Arg Pro Arg Phe Ser Arg His Ser 580 585 590 ThrIle Leu Asp Tyr Ile Asn Val Val Pro Thr Ala Gly Pro Leu Ala 595 600 605Gln Lys Arg Asn Gln Lys Ala Thr Pro Asn Ser Pro Arg Thr Pro Leu 610 615620 Pro Pro Gly Ala Pro Ser Pro Glu Ser Lys Lys Asn Gln Lys Lys Gln 625630 635 640 Tyr Gln Leu Pro Ser Phe Pro Glu Pro Lys Ser Ser Thr Gln AlaPro 645 650 655 Glu Ser Gln Glu Ser Gln Glu Glu Leu His Tyr Ala Thr LeuAsn Phe 660 665 670 Pro Gly Val Arg Pro Arg Pro Glu Ala Arg Met Pro LysGly Thr Gln 675 680 685 Ala Asp Tyr Ala Glu Val Lys Phe Gln 690 695 292529 DNA Artificial Sequence Description of Artificial SequenceSiglec-BMSL3-hIg 29 ccacgcgtcc gggccccagg gctcagcttc cgccttcggcttccccttct gccaagagcc 60 ctgagccact cacagcacga ccagagaaca ggcctgtctcaggcaggccc tgcgcctcct 120 atgcggagat gctactgcca ctgctgctgt cctcgctgctgggcgggtcc caggctatgg 180 atgggagatt ctggatacga gtgcaggagt cagtgatggtgccggagggc ctgtgcatct 240 ctgtgccctg ctctttctcc tacccccgac aagactggacagggtctacc ccagcttatg 300 gctactggtt caaagcagtg actgagacaa ccaagggtgctcctgtggcc acaaaccacc 360 agagtcgaga ggtggaaatg agcacccggg gccgattccagctcactggg gatcccgcca 420 aggggaactg ctccttggtg atcagagacg cgcagatgcaggatgagtca cagtacttct 480 ttcgggtgga gagaggaagc tatgtgagat ataatttcatgaacgatggg ttctttctaa 540 aagtaacagc cctgactcag aagcctgatg tctacatccccgagaccctg gagcccgggc 600 agccggtgac ggtcatctgt gtgtttaact gggcctttgaggaatgtcca cccccttctt 660 tctcctggac gggggctgcc ctctcctccc aaggaaccaaaccaacgacc tcccacttct 720 cagtgctcag cttcacgccc agaccccagg accacaacaccgacctcacc tgccatgtgg 780 acttctccag aaagggtgtg agcgcacaga ggaccgtccgactccgtgtg gcctatgccc 840 ccagagacct tgttatcagc atttcacgtg acaacacgccagccctggag ccccagcccc 900 agggaaatgt cccatacctg gaagcccaaa aaggccagttcctgcggctc ctctgtgctg 960 ctgacagcca gccccctgcc acactgagct gggtcctgcagaacagagtc ctctcctcgt 1020 cccatccctg gggccctaga cccctggggc tggagctgcccggggtgaag gctggggatt 1080 cagggcgcta cacctgccga gcggagaaca ggcttggctcccagcagcga gccctggacc 1140 tctctgtgca gtatcctcca gagaacctga gagtgatggtttcccaagca aacaggacag 1200 tcctggaaaa ccttgggaac ggcacgtctc tcccagtactggagggccaa agcctgtgcc 1260 tggtctgtgt cacacacagc agccccccag ccaggctgagctggacccag aggggacagg 1320 ttctgagccc ctcccagccc tcagaccccg gggtcctggagctgcctcgg gttcaagtgg 1380 agcacgaagg agagttcacc tgccacgctc ggcacccactgggctcccag cacgtctctc 1440 tcagcctctc cgtgcactac tccccgaagc tgctgggcccctcctgctcc tgggaggctg 1500 agggtctgca ctgcagctgc tcctcccagg ccagcccggccccctctctg cgctggtggc 1560 ttggggagga gctgctggag gggaacagca gccaggactccttcgaggtc acccccagct 1620 cagccgggcc ctgggccaac agctccctga gcctccatggagggctcagc tccggcctca 1680 ggctccgctg tgaggcctgg aacgtccatg gggcccagagtggatccatc ctgcagctgc 1740 cagataagaa gggactcatc tcagatccgg agcccaaatcttgtgacaaa actcacacat 1800 gcccaccgtg cccagcacct gaattcgagg gtgcaccgtcagtcttcctc ttccccccaa 1860 aacccaagga caccctcatg atctcccgga cccctgaggtcacatgcgtg gtggtggacg 1920 tgagccacga agaccctgag gtcaagttca actggtacgtggacggcgtg gaggtgcata 1980 atgccaagac aaagccgcgg gaggagcagt acaacagcacgtaccgggtg gtcagcgtcc 2040 tcaccgtcct gcaccaggac tggctgaatg gcaaggagtacaagtgcaag gtctccaaca 2100 aagccctccc agcccccatc gagaaaacca tctccaaagccaaagggcag ccccgagaac 2160 cacaggtgta caccctgccc ccatcccggg atgagctgaccaagaaccag gtcagcctga 2220 cctgcctggt caaaggcttc tatcccagcg acatcgccgtggagtgggag agcaatgggc 2280 agccggagaa caactacaag accacgcctc ccgtgctggactccgacggc tccttcttcc 2340 tctacagcaa gctcaccgtg gacaagagca ggtggcagcaggggaacgtc ttctcatgct 2400 ccgtgatgca tgaggctctg cacaaccact acacgcagaagagcctctcc ctgtctccgg 2460 gtaaatgagt gcgacggccg gcaagccccg ctccccgggctctcgcggtc gcacgaggat 2520 gcttctaga 2529 30 779 PRT Artificial SequenceDescription of Artificial Sequence Siglec-BMSL3-hIg 30 Met Leu Leu ProLeu Leu Leu Ser Ser Leu Leu Gly Gly Ser Gln Ala 1 5 10 15 Met Asp GlyArg Phe Trp Ile Arg Val Gln Glu Ser Val Met Val Pro 20 25 30 Glu Gly LeuCys Ile Ser Val Pro Cys Ser Phe Ser Tyr Pro Arg Gln 35 40 45 Asp Trp ThrGly Ser Thr Pro Ala Tyr Gly Tyr Trp Phe Lys Ala Val 50 55 60 Thr Glu ThrThr Lys Gly Ala Pro Val Ala Thr Asn His Gln Ser Arg 65 70 75 80 Glu ValGlu Met Ser Thr Arg Gly Arg Phe Gln Leu Thr Gly Asp Pro 85 90 95 Ala LysGly Asn Cys Ser Leu Val Ile Arg Asp Ala Gln Met Gln Asp 100 105 110 GluSer Gln Tyr Phe Phe Arg Val Glu Arg Gly Ser Tyr Val Arg Tyr 115 120 125Asn Phe Met Asn Asp Gly Phe Phe Leu Lys Val Thr Ala Leu Thr Gln 130 135140 Lys Pro Asp Val Tyr Ile Pro Glu Thr Leu Glu Pro Gly Gln Pro Val 145150 155 160 Thr Val Ile Cys Val Phe Asn Trp Ala Phe Glu Glu Cys Pro ProPro 165 170 175 Ser Phe Ser Trp Thr Gly Ala Ala Leu Ser Ser Gln Gly ThrLys Pro 180 185 190 Thr Thr Ser His Phe Ser Val Leu Ser Phe Thr Pro ArgPro Gln Asp 195 200 205 His Asn Thr Asp Leu Thr Cys His Val Asp Phe SerArg Lys Gly Val 210 215 220 Ser Ala Gln Arg Thr Val Arg Leu Arg Val AlaTyr Ala Pro Arg Asp 225 230 235 240 Leu Val Ile Ser Ile Ser Arg Asp AsnThr Pro Ala Leu Glu Pro Gln 245 250 255 Pro Gln Gly Asn Val Pro Tyr LeuGlu Ala Gln Lys Gly Gln Phe Leu 260 265 270 Arg Leu Leu Cys Ala Ala AspSer Gln Pro Pro Ala Thr Leu Ser Trp 275 280 285 Val Leu Gln Asn Arg ValLeu Ser Ser Ser His Pro Trp Gly Pro Arg 290 295 300 Pro Leu Gly Leu GluLeu Pro Gly Val Lys Ala Gly Asp Ser Gly Arg 305 310 315 320 Tyr Thr CysArg Ala Glu Asn Arg Leu Gly Ser Gln Gln Arg Ala Leu 325 330 335 Asp LeuSer Val Gln Tyr Pro Pro Glu Asn Leu Arg Val Met Val Ser 340 345 350 GlnAla Asn Arg Thr Val Leu Glu Asn Leu Gly Asn Gly Thr Ser Leu 355 360 365Pro Val Leu Glu Gly Gln Ser Leu Cys Leu Val Cys Val Thr His Ser 370 375380 Ser Pro Pro Ala Arg Leu Ser Trp Thr Gln Arg Gly Gln Val Leu Ser 385390 395 400 Pro Ser Gln Pro Ser Asp Pro Gly Val Leu Glu Leu Pro Arg ValGln 405 410 415 Val Glu His Glu Gly Glu Phe Thr Cys His Ala Arg His ProLeu Gly 420 425 430 Ser Gln His Val Ser Leu Ser Leu Ser Val His Tyr SerPro Lys Leu 435 440 445 Leu Gly Pro Ser Cys Ser Trp Glu Ala Glu Gly LeuHis Cys Ser Cys 450 455 460 Ser Ser Gln Ala Ser Pro Ala Pro Ser Leu ArgTrp Trp Leu Gly Glu 465 470 475 480 Glu Leu Leu Glu Gly Asn Ser Ser GlnAsp Ser Phe Glu Val Thr Pro 485 490 495 Ser Ser Ala Gly Pro Trp Ala AsnSer Ser Leu Ser Leu His Gly Gly 500 505 510 Leu Ser Ser Gly Leu Arg LeuArg Cys Glu Ala Trp Asn Val His Gly 515 520 525 Ala Gln Ser Gly Ser IleLeu Gln Leu Pro Asp Lys Lys Gly Leu Ile 530 535 540 Ser Asp Pro Glu ProLys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 545 550 555 560 Cys Pro AlaPro Glu Phe Glu Gly Ala Pro Ser Val Phe Leu Phe Pro 565 570 575 Pro LysPro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 580 585 590 CysVal Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 595 600 605Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 610 615620 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 625630 635 640 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys ValSer 645 650 655 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser LysAla Lys 660 665 670 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro ProSer Arg Asp 675 680 685 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys LeuVal Lys Gly Phe 690 695 700 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu SerAsn Gly Gln Pro Glu 705 710 715 720 Asn Asn Tyr Lys Thr Thr Pro Pro ValLeu Asp Ser Asp Gly Ser Phe 725 730 735 Phe Leu Tyr Ser Lys Leu Thr ValAsp Lys Ser Arg Trp Gln Gln Gly 740 745 750 Asn Val Phe Ser Cys Ser ValMet His Glu Ala Leu His Asn His Tyr 755 760 765 Thr Gln Lys Ser Leu SerLeu Ser Pro Gly Lys 770 775 31 2052 DNA Artificial Sequence Descriptionof Artificial Sequence Siglec-BMSL3a-hIg 31 ccacgcgtcc gggccccagggctcagcttc cgccttcggc ttccccttct gccaagagcc 60 ctgagccact cacagcacgaccagagaaca ggcctgtctc aggcaggccc tgcgcctcct 120 atgcggagat gctactgccactgctgctgt cctcgctgct gggcgggtcc caggctatgg 180 atgggagatt ctggatacgagtgcaggagt cagtgatggt gccggagggc ctgtgcatct 240 ctgtgccctg ctctttctcctacccccgac aagactggac agggtctacc ccagcttatg 300 gctactggtt caaagcagtgactgagacaa ccaagggtgc tcctgtggcc acaaaccacc 360 agagtcgaga ggtggaaatgagcacccggg gccgattcca gctcactggg gatcccgcca 420 aggggaactg ctccttggtgatcagagacg cgcagatgca ggatgagtca cagtacttct 480 ttcgggtgga gagaggaagctatgtgagat ataatttcat gaacgatggg ttctttctaa 540 aagtaacagt gctcagcttcacgcccagac cccaggacca caacaccgac ctcacctgcc 600 atgtggactt ctccagaaagggtgtgagcg cacagaggac cgtccgactc cgtgtggcct 660 atgcccccag agaccttgttatcagcattt cacgtgacaa cacgccagcc ctggagcccc 720 agccccaggg aaatgtcccatacctggaag cccaaaaagg ccagttcctg cggctcctct 780 gtgctgctga cagccagccccctgccacac tgagctgggt cctgcagaac agagtcctct 840 cctcgtccca tccctggggccctagacccc tggggctgga gctgcccggg gtgaaggctg 900 gggattcagg gcgctacacctgccgagcgg agaacaggct tggctcccag cagcgagccc 960 tggacctctc tgtgcagtatcctccagaga acctgagagt gatggtttcc caagcaaaca 1020 ggacagtcct ggaaaaccttgggaacggca cgtctctccc agtactggag ggccaaagcc 1080 tgtgcctggt ctgtgtcacacacagcagcc ccccagccag gctgagctgg acccagaggg 1140 gacaggttct gagcccctcccagccctcag accccggggt cctggagctg cctcgggttc 1200 aagtggagca cgaaggagagttcacctgcc acgctcggca cccactgggc tcccagcacg 1260 tctctctcag cctctccgtgcactaggatc cggagcccaa atcttgtgac aaaactcaca 1320 catgcccacc gtgcccagcacctgaattcg agggtgcacc gtcagtcttc ctcttccccc 1380 caaaacccaa ggacaccctcatgatctccc ggacccctga ggtcacatgc gtggtggtgg 1440 acgtgagcca cgaagaccctgaggtcaagt tcaactggta cgtggacggc gtggaggtgc 1500 ataatgccaa gacaaagccgcgggaggagc agtacaacag cacgtaccgg gtggtcagcg 1560 tcctcaccgt cctgcaccaggactggctga atggcaagga gtacaagtgc aaggtctcca 1620 acaaagccct cccagcccccatcgagaaaa ccatctccaa agccaaaggg cagccccgag 1680 aaccacaggt gtacaccctgcccccatccc gggatgagct gaccaagaac caggtcagcc 1740 tgacctgcct ggtcaaaggcttctatccca gcgacatcgc cgtggagtgg gagagcaatg 1800 ggcagccgga gaacaactacaagaccacgc ctcccgtgct ggactccgac ggctccttct 1860 tcctctacag caagctcaccgtggacaaga gcaggtggca gcaggggaac gtcttctcat 1920 gctccgtgat gcatgaggctctgcacaacc actacacgca gaagagcctc tccctgtctc 1980 cgggtaaatg agtgcgacggccggcaagcc ccgctccccg ggctctcgcg gtcgcacgag 2040 gatgcttcta ga 2052 32619 PRT Artificial Sequence Description of Artificial SequenceSiglec-BMSL3a-hIg 32 Met Leu Leu Pro Leu Leu Leu Ser Ser Leu Leu Gly GlySer Gln Ala 1 5 10 15 Met Asp Gly Arg Phe Trp Ile Arg Val Gln Glu SerVal Met Val Pro 20 25 30 Glu Gly Leu Cys Ile Ser Val Pro Cys Ser Phe SerTyr Pro Arg Gln 35 40 45 Asp Trp Thr Gly Ser Thr Pro Ala Tyr Gly Tyr TrpPhe Lys Ala Val 50 55 60 Thr Glu Thr Thr Lys Gly Ala Pro Val Ala Thr AsnHis Gln Ser Arg 65 70 75 80 Glu Val Glu Met Ser Thr Arg Gly Arg Phe GlnLeu Thr Gly Asp Pro 85 90 95 Ala Lys Gly Asn Cys Ser Leu Val Ile Arg AspAla Gln Met Gln Asp 100 105 110 Glu Ser Gln Tyr Phe Phe Arg Val Glu ArgGly Ser Tyr Val Arg Tyr 115 120 125 Asn Phe Met Asn Asp Gly Phe Phe LeuLys Val Thr Val Leu Ser Phe 130 135 140 Thr Pro Arg Pro Gln Asp His AsnThr Asp Leu Thr Cys His Val Asp 145 150 155 160 Phe Ser Arg Lys Gly ValSer Ala Gln Arg Thr Val Arg Leu Arg Val 165 170 175 Ala Tyr Ala Pro ArgAsp Leu Val Ile Ser Ile Ser Arg Asp Asn Thr 180 185 190 Pro Ala Leu GluPro Gln Pro Gln Gly Asn Val Pro Tyr Leu Glu Ala 195 200 205 Gln Lys GlyGln Phe Leu Arg Leu Leu Cys Ala Ala Asp Ser Gln Pro 210 215 220 Pro AlaThr Leu Ser Trp Val Leu Gln Asn Arg Val Leu Ser Ser Ser 225 230 235 240His Pro Trp Gly Pro Arg Pro Leu Gly Leu Glu Leu Pro Gly Val Lys 245 250255 Ala Gly Asp Ser Gly Arg Tyr Thr Cys Arg Ala Glu Asn Arg Leu Gly 260265 270 Ser Gln Gln Arg Ala Leu Asp Leu Ser Val Gln Tyr Pro Pro Glu Asn275 280 285 Leu Arg Val Met Val Ser Gln Ala Asn Arg Thr Val Leu Glu AsnLeu 290 295 300 Gly Asn Gly Thr Ser Leu Pro Val Leu Glu Gly Gln Ser LeuCys Leu 305 310 315 320 Val Cys Val Thr His Ser Ser Pro Pro Ala Arg LeuSer Trp Thr Gln 325 330 335 Arg Gly Gln Val Leu Ser Pro Ser Gln Pro SerAsp Pro Gly Val Leu 340 345 350 Glu Leu Pro Arg Val Gln Val Glu His GluGly Glu Phe Thr Cys His 355 360 365 Ala Arg His Pro Leu Gly Ser Gln HisVal Ser Leu Ser Leu Ser Val 370 375 380 His Asp Pro Glu Pro Lys Ser CysAsp Lys Thr His Thr Cys Pro Pro 385 390 395 400 Cys Pro Ala Pro Glu PheGlu Gly Ala Pro Ser Val Phe Leu Phe Pro 405 410 415 Pro Lys Pro Lys AspThr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 420 425 430 Cys Val Val ValAsp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 435 440 445 Trp Tyr ValAsp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 450 455 460 Glu GluGln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 465 470 475 480Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 485 490495 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 500505 510 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp515 520 525 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys GlyPhe 530 535 540 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly GlnPro Glu 545 550 555 560 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp SerAsp Gly Ser Phe 565 570 575 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys SerArg Trp Gln Gln Gly 580 585 590 Asn Val Phe Ser Cys Ser Val Met His GluAla Leu His Asn His Tyr 595 600 605 Thr Gln Lys Ser Leu Ser Leu Ser ProGly Lys 610 615 33 21 DNA Artificial Sequence Description of ArtificialSequence L3 forward primer 33 tgctcagctt cacgcccaga c 21 34 20 DNAArtificial Sequence Description of Artificial Sequence L3 reverse primer34 tgcacggaga ggctgagaga 20 35 19 DNA Artificial Sequence Description ofArtificial Sequence S1 forward primer 35 ctcagaagcc tgatgtcta 19 36 18DNA Artificial Sequence Description of Artificial Sequence S1 reverseprimer 36 gagaagtggg aggtcgtt 18 37 18 DNA Artificial SequenceDescription of Artificial Sequence S2 forward primer 37 ctgctgggcccctcctgc 18 38 19 DNA Artificial Sequence Description of ArtificialSequence S2 reverse primer 38 gacgttccag gcctcacag 19 39 20 DNAArtificial Sequence Description of Artificial Sequence P1 forward primer39 ccttcggctt ccccttctgc 20 40 19 DNA Artificial Sequence Description ofArtificial Sequence P1 reverse primer 40 cgttggtttg gttccttgg 19 41 19DNA Artificial Sequence Description of Artificial Sequence P2 forwardprimer 41 cacactgagc tgggtcctg 19 42 19 DNA Artificial SequenceDescription of Artificial Sequence P2 reverse primer 42 gacgttccaggcctcacag 19 43 19 DNA Artificial Sequence Description of ArtificialSequence P3 forward primer 43 cacactgagc tgggtcctg 19 44 20 DNAArtificial Sequence Description of Artificial Sequence P3 reverse primer44 gaaaagaaga gccgtgatgc 20 45 18 DNA Artificial Sequence Description ofArtificial Sequence L3-TM forward primer 45 tgcagctgcc agataaga 18 46 18DNA Artificial Sequence Description of Artificial Sequence L3-TM reverseprimer 46 ggcttgagtg gatgattt 18 47 19 DNA Artificial SequenceDescription of Artificial Sequence S1 forward primer 47 ctccgaagcctgatgtcta 19 48 18 DNA Artificial Sequence Description of ArtificialSequence S1 reverse primer 48 gagaagtggg aggtcgtt 18 49 18 DNAArtificial Sequence Description of Artificial Sequence S2 forward primer49 ctgctgggcc cctcctgc 18 50 19 DNA Artificial Sequence Description ofArtificial Sequence S2 reverse primer 50 gacgttccag gcctcacag 19 51 20DNA Artificial Sequence Description of Artificial Sequence Beta-actinforward primer 51 gtggggcgcc ccaggcacca 20 52 23 DNA Artificial SequenceDescription of Artificial Sequence Beta-actin reverse primer 52ctccttaatg tcacgcacga ttc 23 53 18 DNA Artificial Sequence Descriptionof Artificial Sequence L3-TM forward primer 53 tgcagctgcc agataaga 18 5418 DNA Artificial Sequence Description of Artificial Sequence L3-TMreverse primer 54 ggcttgagtg gatgattt 18 55 36 DNA Artificial SequenceDescription of Artificial Sequence GST-SiglecBMSL3cyto (wt) forwardprimer 55 gcggccagga attccaagag acggactcag acagaa 36 56 33 DNAArtificial Sequence Description of Artificial SequenceGST-SiglecBMSL3cyto (wt) reverse primer 56 gcggccctcg agtcattggaacttgacttc tgc 33 57 57 DNA Artificial Sequence Description ofArtificial Sequence GST-SiglecBMSL3Y641F forward primer 57 ccagaatcaaagaagaacca gaaaaagcag tttcagttgc ccagtttccc agaaccc 57 58 56 DNAArtificial Sequence Description of Artificial SequenceGST-SiglecBMSL3Y641F reverse primer 58 gggttctggg aaactgggca actgaactgctttttctggt tcttctttga ttctgg 56 59 45 DNA Artificial SequenceDescription of Artificial Sequence GST-SiglecBMSL3Y667F forward primer59 gagagccaag aggagctcca ttttgccacg ctcaacttcc caggc 45 60 45 DNAArtificial Sequence Description of Artificial SequenceGST-SiglecBMSL3Y667F reverse primer 60 gcctgggaag ttgagcgtgg caaaatggagctcctcttgg ctctc 45 61 51 DNA Artificial Sequence Description ofArtificial Sequence GST-SiglecBMSL3Y691F forward primer 61 gcggccctcgagtcattgga acttgacttc tgcaaaatcc gcctgggtgc c 51 62 51 DNA ArtificialSequence Description of Artificial Sequence GST-SiglecBMSL3Y691F reverseprimer 62 gcggccctcg agtcattgga acttgacttc tgcaaaatcc gcctgggtgc c 51 6339 DNA Artificial Sequence Description of Artificial SequenceGST-SiglecBMSL3Y641 alone forward primer 63 gcggccagga attccatcaatgtggtcccg acggctggc 39 64 36 DNA Artificial Sequence Description ofArtificial Sequence GST-SiglecBMSL3Y641 alone reverse primer 64gcggccctcg agtcaatgga gctcctcttg gctctc 36 65 109 DNA ArtificialSequence Description of Artificial Sequence SiglecBMSL3a hIg forwardprimer 65 ccgcctaagc tttccccttc tgccaagagc cctgagccct gagccactcacagcacgacc 60 agagaacagg cctgtctcag gcaggccctg cgcctcctat gcggagatg 10966 35 DNA Artificial Sequence Description of Artificial SequenceSiglecBMSL3a hIg reverse primer 66 gaagatctga accatggtta tagtgcacggagagg 35 67 109 DNA Artificial Sequence Description of ArtificialSequence SiglecBMSL3 hIg forward primer 67 ccgcctaagc tttccccttctgccaagagc cctgagccct gagccactca cagcacgacc 60 agagaacagg cctgtctcaggcaggccctg cgcctcctat gcggagatg 109 68 35 DNA Artificial SequenceDescription of Artificial Sequence SiglecBMSL3 hIg reverse primer 68gaagatctga accatggtta ggagaatgcc gttga 35 69 241 DNA Artificial SequenceDescription of Artificial Sequence partial 5′ sequence of 3421048 69caggcctgtc tcaggcaggc cctgcgcctc ctatgcggag atgctactgc cactgctgct 60gtcctcgctg ctgggcgggt cccangctat ggatgggaga ttctggatac gagtgcagga 120gtcagtgatg gtgccggagg gcctgtgcat ctctgtgccc tgctctttct cctacccccg 180acaggactgg acagggtcta ccccagctta tggctactgg ttcaaagcag tgactgagac 240 a241 70 21 DNA Artificial Sequence Description of Artificial SequenceSiglec 10 L3 probe 70 tgctcagctt cacgcccaga c 21 71 20 DNA ArtificialSequence Description of Artificial Sequence Siglec 10 L3 probe 71tgcacggaga ggctgagaga 20

What is claimed is:
 1. An isolated SIGLEC protein comprising an aminoacid sequence beginning with Ala141 and ending with Ser198 as shown inFIG. 6B.
 2. An isolated SIGLEC protein comprising an amino acid sequencebeginning with Ala141 and ending with Ser198 as shown in FIG. 6B and isencoded by a nucleic acid molecule that hybridizes, under stringentconditions to a nucleic acid molecule that is complementary to thenucleic acid as shown in any one of FIGS. 2A, 3A, 4A, 5A, 6A, 7A, 8A,and 9A.
 3. The isolated SIGLEC protein of claim 1 or 2 comprising theamino acid sequences as shown in any one of FIGS. 4B, 5B, and 6B.
 4. Anisolated SIGLEC protein comprising an amino acid sequence that isencoded by a nucleic acid molecule that hybridizes, under stringentconditions to a nucleic acid molecule that is complementary to thenucleic acid as shown in any one of FIGS. 2A, 3A, 4A, 5A, 6A, 7A, 8A,and 9A.
 5. An isolated SIGLEC protein of claim 4 having an amino acidsequence as shown in any of FIGS. 2B, 3B, 4B, 5B, 6B, 7B, 8B.
 6. Apeptide fragment of the protein of claim
 3. 7. A peptide fragment ofclaim 6 having an amino acid sequence beginning with Ala14l and endingwith Ser198 as shown in FIG. 6B, or any fragment thereof.
 8. A peptidefragment of claim 6 comprising the cytoplasmic domain having an aminoacid sequence beginning with Lys576 and ending with Gln697 as shown inFIG. 6B or any fragment thereof.
 9. A mutant SIGLEC BMS proteincomprising a cytoplasmic domain, wherein at least one tyrosine in thecytoplasmic domain is substituted with an amino acid selected from thegroup consisting of phenylalanine, leucine, tryptophan, and threonine.10. The mutant SIGLEC BMS protein of claim 9 having the amino acidsshown in FIG. 6b, wherein the tyrosine in the cytoplasmic domain is anyof the tyrosines at position 597, 641, 667, or
 691. 11. An isolatedSiglec nucleic acid molecule comprising a nucleic acid beginning withcodon GCC at position +421 and ending at codon TCA at position +594 asshown in FIG. 6A, wherein the nucleic acid hybridizes, under stringentconditions to a nucleic acid molecule that is complementary to thenucleic acid as shown in any one of FIGS. 2A, 3A, 4A, 5A, 6A, 7A, 8A,and 9A.
 12. An isolated Siglec nucleic acid molecule that encodes theprotein of claim 1, 2, or
 4. 13. The isolated Siglec nucleic acidmolecule of claim 12, comprising the sequence shown in any of (a) FIG.2A beginning at codon GGC at position +12 and ending at codon CCA atposition +1760; (b) FIG. 3A beginning at codon GAT at position +3 andending at codon CAA at position +1868; (c) FIG. 4A beginning at codonGGA at position +12 and ending at codon CAA at position +1736; (d) FIG.5A beginning at codon CCC at position +2 and ending at codon ATG atposition +1291; (e) FIG. 6A beginning at codon ATG at position +1 andending at codon CAA at position +2091; (f) FIG. 7A beginning at codonCTG at position +1 and ending at codon GGC at position +1398; (g) FIG.8A beginning at codon ATG at position +43 and ending at codon AGA atposition +1431; or (h) FIG. 9A beginning at codon ATG at position +57and ending at codon AGT at position +914.
 14. The isolated Siglecnucleic acid molecule of claim 11, or 13, which is DNA or RNA.
 15. Anisolated nucleic acid molecule which is complementary to the nucleotidesequence of the molecule of claim 11, or
 13. 16. A vector comprising theisolated nucleic acid molecule of claim 11, or
 13. 17. A host-vectorsystem comprising the vector of claim 16, in a suitable host cell.
 18. Amethod for producing a SIGLEC-BMS protein having the amino acid sequenceof any of FIGS. 2B, 3B, 4B, 5B, 6B, 7B, 8B, and 9B, comprising: a)culturing the host-vector system of claim 17 under suitable conditionsso as to produce the protein; and b) recovering the protein so produced.19. A SIGLEC-BMS protein produced by the method of claim
 18. 20. Achimeric protein comprising the polypeptide of claim 1, 2, or 4, or afragment thereof, fused to a heterologous polypeptide.
 21. A chimericprotein comprising an extracellular domain of the polypeptide of claim1, 2, or 4, fused to a heterologous polypeptide.
 22. A chimeric proteincomprising the cytoplasmic domain of the polypeptide of claim 1, 2, or4, fused to a heterologous polypeptide.
 23. The chimeric protein ofclaim 20, 21, or 22, wherein the heterologous polypeptide is animmunoglobulin constant region.
 24. The chimeric fusion protein of claim20, 21, or 22, wherein the heterologous protein is GlutathioneS-transferase.
 25. An antibody or antibody fragment having an antigenbinding site, wherein the antigen binding site specifically recognizesand binds the protein of claim 1, 2 or
 4. 26. The antibody of claim 25,wherein the antibody is a polyclonal antibody or a monoclonal antibody.27. The antibody of claim 26, wherein the monoclonal antibody isdesignated SIGLEC-10-9, SIGLEC-10-13, SIGLEC-10-14, SIGLEC-10-27, orSIGLEC-10-61, and which are collectively deposited as ATCC Accession No(______).
 28. The antibody of claim 25, wherein the antibody is achimeric antibody having a murine antigen-binding site and a humanizedregion that regulates effector functions.
 29. A method for identifying atest molecule that modulates an immune response induced by Siglec-10positive cells comprising: a. contacting Siglec-10 positive cells withthe test molecule; and b. determining whether the immune response ismodulated.
 30. The method of claim 29, wherein the test molecule thatmodulates an immune response targets an extracellular domain of aSiglec-10 on Siglec-10 positive cells.
 31. The method of claim 30,wherein the extracellular domain encompasses at least one of the Ig-likedomains of a Siglec-10.
 32. The method of claim 31, wherein the Ig-likedomain of Siglec-10 ia an Ig (V) domain of a Siglec-10.
 33. The methodof claim 31, wherein the Ig-like domain of Siglec-10 is an Ig (C) domainof a Siglec-10.
 34. A method for modulating an immune response inducedby Siglec-10 positive cells comprising contacting Siglec-10 positivecells with a monoclonal antibody directed against Siglec-10 undersuitable conditions so that the immune response is modulated.
 35. Themethod of claim 34, wherein the antibody targets an extracellular domainof a Siglec-10.
 36. The method of claim 35, wherein the extracellulardomain so targeted is an Ig-like domain of a Siglec-10.
 37. The methodof claim 36, wherein the Ig-like domain is an Ig (V) domain of aSiglec-10.
 38. The method of claim 36, wherein the Ig-like domain is anIg (C) domain of a Siglec-10.